Transglutaminase inhibitors and methods of use thereof

ABSTRACT

Transglutaminase inhibitors and methods of use thereof are provided.

GOVERNMENT SUPPORT

This invention was made with Government support under contract DK063158awarded by the National Institutes of Health. The Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

Transglutaminases belong to a family of enzymes that play importantroles in diverse biological functions by selectively cross-linkingproteins. They catalyze formation of ε-(γ-glutamyl)-lysine cross-linksbetween proteins, and may also incorporate polyamines into suitableprotein substrates. This covalent isopeptide cross-link is stable andresistant to proteolysis, thereby increasing the resistance of tissue tochemical, enzymatic, and mechanical disruption. Among the members ofthis family are plasma transglutaminase, factor XIIIa, which stabilizesfibrin clots; keratinocyte transglutaminase and epidermaltransglutaminase, which cross-link proteins on the outer surface ofsquamous epithelia; and tissue transglutaminase, which cross-linksfibronectin in the extracellular matrix of organs such as brain, liverand the intestine.

Transglutaminase 2 (TG2, also known as tissue transglutaminase), acalcium-dependent member of the transglutaminase family, is reported tohave extracellular as well as intracellular functions. Outside the cellTG2 plays a crucial role in shaping the extracellular matrix bycross-linking fibronectin and related proteins. TG2 also promotes celladhesion and motility by forming non-covalent complexes with other keyproteins such as integrins and fibronectin. Intracellular TG2 losesenzyme activity when bound to GTP, but functions as a G-protein in thephospholipase C signal transduction cascade. Human TG2 is a structurallyand mechanistically complex protein. Its catalytic mechanism is similarto that employed by cysteine proteases, involving a catalytic triad ofcysteine, histidine, and aspartate. The cysteine thiol group reacts witha glutamine sidechain of a protein substrate to form a reactivethioester intermediate, from which the acyl group is transferred toanother amine substrate.

Several members of the transglutaminase family have been linked todisease, including tissue transglutaminase (TG2), and the skintransglutaminases, TG1 and TG3. TG2 is a cytoplasmic enzyme present inmany cells, including those in the blood vessel wall. Aberrant TG2activity is believed to play a role in neurological disorders such asAlzheimer's, Parkinson's and Huntington's disease (see, for example, Kimet al. (2002) Neurochem. Int. 40:85-103; Karpuj et al. (2002) NatureMed. 8, 143-149). In Celiac Sprue, where TG2 is the predominantautoantigen, its pivotal role in unmasking antigenic epitopes by sitespecific deamidation of gluten peptides is well established. Expressionof TG1 and TG2 have been correlated with various types of malignancies(see Zhang et al. (2003) Glia 42:194-208; and Martinet et al. (2993) Am.J. Respir. Cell. Mol. Biol. 28, 428-435), including glioblastomas, lungand breast cancers, suggesting an important role for TG2 in tumorproliferation and survival. Taken together, the above findings make astrong case for suitable small molecule TG2 inhibitors as experimentaltherapeutic agents. The medicinal attractiveness of this protein targetis underscored by the observation that TG2 knockout mice are normal,lacking developmental, physiological or reproductive defects.

Although a number of TG2 inhibitors have been used in biological studiesover the past two decades, many of these compounds (e.g. monodansylcadaverine) contain primary amines in addition to potential inhibitorymotifs, and it remains unclear whether the observed effects are due toexcess competing amines or by blockage of TG2 substrate turnover. A fewstudies have utilized a suicide inhibitor, L682777, which inhibits humanTG2 (Lorand et al. (1998) Exp Eye Res. 66:531-6). However, L682777 wasdesigned as a specific inhibitor of Factor XIIIa, and is thereforeunsuitable for evaluating TG2 biology in vivo. More recently,mechanism-based active-site inhibitors of guinea pig and human (Hauschet al. (2003) Chem Biol 10, 225-231; Choi et al. (2005) Chem. Biol. 12,469-475) TG2 have been reported.

In view of the serious and widespread nature of Celiac Sprue and thedifficulty of removing gluten from the diet, better methods of treatmentare of great interest. In particular, there is a need for treatmentmethods that allow the Celiac Sprue individual to eat gluten-containingfoodstuffs without ill effect or at least to tolerate such foodstuffs insmall or moderate quantities without inducing relapse. The presentinvention meets this need for better therapies for Celiac Sprue byproviding new drugs and methods and formulations of new and existingdrugs to treat Celiac Sprue. International Patent ApplicationUS03/04743, herein specifically incorporated by reference, disclosesaspects of gluten protease stability and immunogenicity.

tTGase has also been implicated in certain cancers. Neuro-oncologicaldiseases including malignant neoplasms such as glioblastomas andmelanomas metastatic to the brain are notoriously resistant to standardradiation and chemotherapy treatment. Current treatment strategiesgenerally fail to achieve long-term survival. Similarly certain benignCNS tumors such as meningiomas are resistant to chemotherapy andradiation. Current treatment strategies with these tumors typicallyrequire major surgical resections or treatment with radiation in anattempt to control growth of recurrent or non-resectable tumors.Meningiomas are generally resistant to radiation-induced cell death andto chemotherapy. The mechanisms responsible for the failure of thesebrain tumors to respond to chemotherapy and radiation are not known.Therefore, identification of agents that augment sensitivity tochemotherapy and radiation therapy is important for improving treatmentstrategies in patients with these and other refractory cancers.

SUMMARY OF THE INVENTION

The present invention provides methods that utilize administration of atransglutaminase inhibitor to a patient for the treatment of conditionsassociated with undesirable transglutaminase activity. The inhibition oftissue transglutaminase (tTGase; TG2) is of particular interest. tTGaseinhibitors of interest include small molecule tTGase inhibitorscomprising a 3-halo-4,5-dihydroisoxazole moiety, e.g. inhibitorscomprising 3-bromo-4,5-dihydroisoxazole; etc. In some embodiments, thedihydroisoxazole moiety is present as a racemic mixture. In otherembodiments, the dihydroisoxazole moiety is present as a substantiallypure (R) or (S) enantiomer.

In one embodiment, the present invention provides novel derivativecompounds of 3-halo-4,5-dihydroisoxazoles and methods of treatment byadministering those compounds. In another embodiment, the tTGaseinhibitor employed in the method is an analog of isatin (2,3diketoindoline).

In another embodiment, the invention provides pharmaceuticalformulations comprising a tTGase inhibitor and a pharmaceuticallyacceptable carrier. In certain embodiments, the formulation alsocomprises one or more glutenases, as described in U.S. ProvisionalApplication 60/392,782 filed Jun. 28, 2002; and U.S. ProvisionalApplication 60/428,033, filed Nov. 20, 2002, both of which areincorporated herein by reference. In other embodiments, the formulationcomprises a chemotherapeutic agent.

The invention also provides methods for the administration of entericformulations of one or more tTGase inhibitors to treat Celiac Sprue.

In another aspect, the tTGase inhibitors and/or pharmaceuticalformulations of the present invention are useful in treating cancer,including neurologic cancers, such as gliomas, astrocytomas, meningiomas(which are cancers of neural crest-derived cells), etc., and othercancers, including melanoma, as well as other neurological disordersincluding Alzheimer's and Huntington's diseases, where the TGases appearto be a factor in the formation of inappropriate proteinaceousaggregates. The tTGase inhibitors act on some cancers to sensitize thetumor cells to killing by chemotherapeutic agents and/or radiation.

These and other aspects and embodiments of the invention and methods formaking and using the invention are described in more detail in thedescription of the drawings and the invention, the examples, the claims,and the drawings that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Comparison of immunohistochemical staining of fibronectin intumors from BCNU-treated mouse brains (right) and from mice treated withBCNU+35 mg/kg KCC009 (left).

FIG. 2: KCC009 as a radiosensitizer. Cultured DBT cells were treatedwith radiation alone (3.2 Gy) (left) or radiation plus 0.5 mM KCC009(right), and plated on soft agar to evaluate cell viability. Atcorresponding concentrations, KCC009 alone does not affect cellviability.

FIG. 3: TG2 activities of the 30 brain tumors and 4 non-malignant braintissue samples

FIG. 4: Top left: control IOMM meningioma cells; Top right: IOMM cellsexposed to 20 Gy radiation; Bottom left: IOMM cells exposed to 0.5 mMKCC009 and 20 Gy radiation.

FIG. 5: Western blot analysis of lysates from IOMM cells treated withKCC009 and/or BCNU. Lysates were immunoprecipitated withanti-fibronectin antibodies, and the Western blots were probed withanti-TG2 antibodies. Lanes (left to right): Control, KCC009 only, BCNUonly, KCC009+BCNU. KCC009 and BCNU were used at concentrations of 0.5 mMand 6.25 μM, respectively. Disappearance of TG2 from lanes treated withKCC009 illustrates loss of association of TG2 with the extracellularmatrix.

FIG. 6A-6D: Photomicrographs of B16 (mouse melanoma cells) after 24 htreatment with KCC009 and/or BCNU. A) Control; B) 1 mM KCC009 only; C)6.25 μM BCNU only; D) KCC009+BCNU combination.

FIG. 7: Time dependent inhibition of small intestine mucosal TG2 in micedosed intra-peritoneally with 60 mg/kg KCC009. Mice were sacrificed atdifferent times, intestinal mucosa were harvested and assayed for TG2activity. The activity of TG2 in jejunal tissue from TG2-knockout miceis <5% of the observed wild-type levels, suggesting the assay employedby us is adequately specific for TG2 activity.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Transglutaminase inhibitors of the invention are administered to apatient for the treatment of conditions associated with undesirabletransglutaminase activity, including Celiac Sprue, dermatitisherpetiformis, cancer, and neurological disorders including Alzheimer'sand Huntington's diseases.

In some embodiments, particularly for the treatment of Celiac Sprue, thecompositions of the invention include formulations of tTGase inhibitorsthat comprise an enteric coating that allows delivery of the agents tothe intestine in an active form; the agents are stabilized to resistdigestion or alternative chemical transformations in acidic stomachconditions. In another embodiment, food is pretreated or combined withglutenase, or a glutenase is co-administered (whether in time or in aformulation of the invention) with a tTGase inhibitor of the invention.

For the treatment of cancer, the tTGase inhibitors can act as asensitizing agent, which enhance killing by a second agent, e.g.radiation, cytotoxic drugs, and the like. For sensitization, the tTGaseinhibitor may be administered separately or in a co-formulation with acytotoxic agent. Although the cytotoxic agents can be active whenadministered alone, the concentrations required for a therapeutic dosemay create undesirable side effects. The combination therapy may providefor a therapeutic effect with less toxicity. Surprisingly, it is shownherein that an (R) enantiomer of a dihydroisoxazole moiety provides forgreater activity against cancer cells than the (S) enantiomer. In someembodiments, a tTGase inhibitor comprising a substantially enantiopure(R) form of a dihydroisoxazole moiety is administered for the treatmentof cancer, and may be administered as a single agent, or in combinationwith a second agent. In other embodiments, a tTGase inhibitor comprisinga substantially enantiopure (S) form of a dihydroisoxazole moiety isadministered for conditions other than cancer, e.g. in the treatment ofCeliac Sprue, dermatitis herpetiformis, and the like.

Methods are provided for the synthesis of pure enantiomers of the tTGasecompounds described herein. Using methods as set forth in the Examples,enantiopure dihydroisoxazole moieties are synthesized, and used toproduce enantiopure compounds, which forms may find use in the methodsof the invention. The tTGase inhibitors, or their pharmaceuticallyacceptable salts may contain one or more asymmetric centers and may thusgive rise to enantiomers, diastereomers, and other stereoisomeric formsthat may be defined, in terms of absolute stereochemistry, as (R)- or(S)- or, as (D)- or (L)- for amino acids. The present invention is meantto include all such possible isomers, as well as, their racemic andoptically pure forms. Optically active (+) and (−), (R)- and (S)-, or(D)- and (L)-isomers may be prepared using chiral synthons or chiralreagents, or resolved using conventional techniques, such as reversephase HPLC. When the compounds described herein contain olefinic doublebonds or other centers of geometric asymmetry, and unless specifiedotherwise, it is intended that the compounds include both E and Zgeometric isomers. Likewise, all tautomeric forms are also intended tobe included.

As used herein, the term “substantially enantiopure” refers tocompositions in which at least about 75% of the compound of interest ispresent as a single stereoisomer, and may refer to compositions in whicha single stereoisomer is at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 99% or more of thecompound of interest. It will be understood by one of skill in the artthat a compound may have multiple asymmetric centers, and that anoptically active stereoisomer may reflect one or more of such asymmetriccenters.

Substantially enantiopure compounds of the invention include, withoutlimitation, molecules comprising (R) and (S) forms of a dihydroisoxazolemoiety, e.g.(S)-[3-(4-hydroxyphenyl)-2-N-(phenylmethyloxycarbonyl)aminopropanoicacid N′-(3′-bromo-5-(S-)-4′,5′-dihydro-5′-isoxalyl)methylamide);(S)-[3-(4-hydroxyphenyl)-2-N-(phenylmethyloxycarbonyl)aminopropanoicacid N′-(3′-bromo-5-(R-)-4′,5′-dihydro-5′-isoxalyl)methylamide);[1-[(3-Bromo-(R)-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(5-fluoro-1H-indol-3-yl)-ethyl]-carbamicacid quinolin-3-ylmethyl ester;[1-[(3-Bromo-(S)-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(5-fluoro-1H-indol-3-yl)-ethyl]-carbamicacid quinolin-3-ylmethyl ester;[1-[(3-Bromo-(R)-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(4-hydroxy-phenyl)-ethyl]-carbamicacid quinolin-3-ylmethyl ester;[1-[(3-Bromo-(S)-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(4-hydroxy-phenyl)-ethyl]-carbamicacid quinolin-3-ylmethyl ester;2-[(3-Bromo-(R)-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carboxylicacid quinolin-3-ylmethyl ester; and2-[(3-Bromo-(S)-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carboxylicacid quinolin-3-ylmethyl ester.

The subject methods are useful for both prophylactic and therapeuticpurposes. Thus, as used herein, the term “treating” is used to refer toboth prevention of disease, and treatment of a pre-existing condition.The treatment of ongoing disease, to stabilize or improve the clinicalsymptoms of the patient, is a particularly important benefit provided bythe present invention. Such treatment is desirably performed prior toloss of function in the affected tissues; consequently, the prophylactictherapeutic benefits provided by the invention are also important. Forexample, treatment of a cancer patient may be reduction of tumor size,elimination of malignant cells, prevention of metastasis, or theprevention of relapse in a patient who has been cured.

Disease Conditions

A number of pathological conditions have been associated withundesirable activity of tissue transglutaminases. For the most part, thedisease-associated transglutaminases of interest for the presentinvention are TG1, TG2, and TG3. Conditions may involve over-expressionof the transglutaminase, expression of transglutaminase in tissues orsites where it is not normally expressed, or may involve normalexpression of transglutaminase in a disease context.

Diseases associated with transglutaminase include, inter alia, celiacsprue; dermatitis herpetiformis; inclusion body myositis;atherosclerosis; Alzheimer's disease; Huntington's disease; Parkinson'sdisease; progressive supranuclear palsy; and tumors, e.g. glioblastomas,meningioma, melanoma, etc.

Celiac Sprue is characterized by damage to the upper small intestine,causing effacement of the villi to produce a characteristically flatmucosa with markedly hypertrophic crypts. Clinical symptoms of CeliacSprue include fatigue, chronic diarrhea, malabsorption of nutrients,weight loss, abdominal distension, anemia, as well as a substantiallyenhanced risk for the development of osteoporosis and intestinalmalignancies (lymphoma and carcinoma). The disease has an incidence ofapproximately 1 in 200 in European populations. Therapeutic effect forCeliac Sprue is measured in terms of clinical outcome, or byimmunological or biochemical tests. Suppression of the deleteriousT-cell activity can be measured by enumeration of reactive Th1 cells, byquantitating the release of cytokines at the sites of lesions, or usingother assays for the presence of autoimmune T cells known in the art.Also both the physician and patient can identify a reduction in symptomsof a disease. Evidence of therapeutic effect may be any diminution inthe severity of disease, particularly diminution of the severity of suchsymptoms as fatigue, chronic diarrhea, malabsorption of nutrients,weight loss, abdominal distension, and anemia. Other disease indiciainclude the presence of antibodies specific for glutens, antibodiesspecific for tissue transglutaminase, the presence of pro-inflammatory Tcells and cytokines, and degradation of the villus structure of thesmall intestine. Application of the methods and compositions of theinvention can result in the improvement of any and all of these diseaseindicia of Celiac Sprue. Patients that can benefit from the presentinvention include both adults and children. Children in particularbenefit from prophylactic treatment, as prevention of early exposure totoxic gluten peptides can prevent development of the disease into itsmore severe forms. Children suitable for prophylaxis in accordance withthe methods of the invention can be identified by genetic testing forpredisposition, e.g. by HLA typing; by family history, and by othermethods known in the art. As is known in the art for other medications,and in accordance with the teachings herein, dosages of the tTGaseinhibitors of the invention can be adjusted for pediatric use.

A related disease is dermatitis herpetiformis, which is a chroniceruption characterized by clusters of intensely pruritic vesicles,papules, and urticaria-like lesions. IgA deposits occur in almost allnormal-appearing and perilesional skin. Asymptomatic gluten-sensitiveenteropathy is found in 75 to 90% of patients and in some of theirrelatives. Onset is usually gradual. Itching and burning are severe, andscratching often obscures the primary lesions with eczematization ofnearby skin, leading to an erroneous diagnosis of eczema.

Gluten proteins, which may include gliadins, secalins and hordeins,contain several sequences rich in Pro-Gln residues that arehigh-affinity substrates for TG2. TG2 is thought to be responsible forgenerating neoepitopes of gluten peptides through deamidation ofglutamine residues. Presentation of these deamidated epitopes by DQ2positive antigen presenting cells effectively stimulates proliferationof gliadin-specific T cells from intestinal biopsies of most CeliacSprue patients. The toxic effects of gluten include immunogenicity ofthe gluten oligopeptides, leading to inflammation. TG2 also cross-linksitself onto gliadin in vitro, and the cross-linked TGase might act as ahapten for the formation of antibodies against gluten peptides.

In addition to these conditions, it has been suggested thattransglutaminase generates autoantibodies in a variety of autoimmunedisorders by cross-linking potential autoantigens and acting as ahapten, e.g. in lupus, myasthenia gravis, multiple sclerosis, rheumatoidarthritis, etc. Anti-TG2 antibodies have been reported in lupus; and TG2has been detected in the synovial fluid of arthritis patients, and theserum and cerebral spinal fluid of amyotrophic lateral sclerosispatients (see review by Kim et al. (2002) Neurochemistry International40:85-103).

Inclusion body myositis is a progressive muscle disorder that affectsolder individuals. It is considered to be an autoimmune disease;associated with the expression of specific HLA molecules and a varietyof autoantibodies. This disease is characterized by a progressivelyworsening weakness in the proximal and distal limbs that is resistant tosteroid therapy. The histological features of this disease includedistinctive rimmed vacuoles and filamentous inclusions, as well asmononuclear infiltrates, which consist predominantly of cytotoxic Tcells. Total transglutaminase enzyme activity is elevated by 16-fold indiseased tissue, which is reflected in an increased number of ε(γ-glutamyl)lysine cross-links. This activity is apparently due to theincreased expression of TG1 and TG2, which co-localize with deposits invacuolated muscle fibers from patients with inclusion body myositis. Thechronic inflammation that characterizes inclusion body myositis may alsostimulate the expression of TG1 and TG2 and thereby contribute to theprogressive nature of this disease.

In addition to autoimmune diseases, transglutaminases are associatedwith neurological conditions. Alzheimer's disease is associated with theselective loss of neurons in the neocortex, hippocampus, and amygdala,resulting in an impaired cognitive ability. This disease is alsocharacterized by the presence of two types of protein aggregates:extracellular neuritic senile plaques, and intraneuronal neurofibrillarytangles. TGases are likely to contribute to the formation of theseaggregates. Isoforms of TGase co-localize with the plaques and tanglesin the brains of Alzheimer's disease patients and the number of ε(γ-glutamyl)lysine linkages in insoluble proteins from Alzheimer'sdisease brains is 30-50 times greater than the number found in normalbrain tissues. In addition, total TGase activity is elevated in theaffected areas of Alzheimer's disease brains, particularly TG1 and TG2.The components of plaques and tangles are substrates for TGases. Forexample, β amyloid, which is found in a fibrillular form in plaques, isa substrate for TGases. Tau, the major component of the paired helicalfilaments that make up neurofibrillary tangles is also a substrate forTG2.

Huntington's disease is characterized by progressive motor andpsychiatric disorders, as well as dementia. The most commonmanifestation of this disease is chorea: involuntary and non-directedmotions that disrupt all normal activities eventually leading to death.The clinical progression of Huntington's disease is accompanied byspecific neuronal loss and dysfunction, particularly in the striatum andlater in the cerebral cortex. Huntington's disease is an autosomaldominant disease. The mutated gene and its product have been identified,and the protein found to be a substrate for TG2. Pathological mutationsof the huntingtin gene, involving expansion of CAG repeats, result instretches of polyglutamines of greater than 39 contiguous glutamineresidues. The age of disease onset correlates inversely with the lengthof the polyglutamine expansion beyond the normal range, and there is adecrease in the age of disease onset with succeeding generations. Thereis elevated transglutaminase activity in the affected regions ofdiseased brains, including the striata. TG1, TG2 and TG3 are present inhuman brains, and all are elevated in Huntington's disease patientbrains.

Parkinson's disease is characterized by tremor, bradykinesia, rigidityand postural instability. These motor disorders result primarily from aloss of dopaminergic neurons of the nigro-striatal pathway.Histologically, Parkinson's disease is characterized by a widespreaddistribution of Lewy bodies, which are intracytoplasmic aggregates ofbetween 5 and 25 mm in diameter that feature a dense eosinophilic coreand pale surrounding halo. Although Lewy bodies are thought to play acausative role in Parkinson's disease, these structures also have beenidentified in some cases of Alzheimer's disease. The major component ofLewy bodies is α-synuclein. A fragment of α-synuclein, known as thenon-amyloid component (NAC), has been detected in the Lewy bodies ofParkinson's disease patients and the neuritic plaques of Alzheimer'sdisease patient. NAC is also as a substrate for transglutaminases, andis neurotoxic to primary dopaminergic neurons, as well as toneuroblastoma cells. TGases catalyze the formation of NAC polymers andaggregates of NAC and β-amyloid peptides.

Progressive supranuclear palsy is a motor disorder that initiallypresents in patients having unexpected falls. The later manifestationsof the disease present as postural instability, vertical gaze palsy,axial rigidity, dysarthria and dementia. The most common pathologicalfindings in this disease are midbrain atrophy with dilation of theaqueduct of Sylvius and depigmentation of the substantia nigra. Thesefeatures are associated with the loss of neurons in the substantianigra, globus pallidus, subthalamic nucleus, basal ganglia, diencephalonand brain stem. Neurofibrillary tangles occur in the affected areas ofprogressive supranuclear palsy patients. Tau, which is a substrate fortransglutaminase, has also been identified as a major component of thedetergent-insoluble proteins isolated from the brains of progressivesupranuclear palsy patients. These aggregates also contain ε(γ-glutamyl)lysine cross links. Expression of TG1 and TG2, particularlyTG1, are elevated in both the cerebellum of progressive supranuclearpalsy patients.

Expression of transglutaminase has also been implicated in certaincancers, including glioblastoma, lung cancer, and cervical cancer.Hilton et al. (1997) Neuropathol Appl Neurobiol. 23(6):507-11 foundexpression of tissue transglutaminase in fibrillary astrocytomas,anaplastic astrocytomas and glioblastomas. Labeling was particularlyprominent in the pseudopalisading tumour cells that surrounded foci ofnecrosis and apoptosis in glioblastomas. Strong transglutaminaselabeling was also observed in the endothelial cells of vessels showingmicrovascular proliferation in all of the glioblastomas studied.Enhanced expression of transglutaminase by endothelial cells inglioblastomas may contribute to the high prevalence of vascularthrombosis and necrosis in these tumours. In cervical cancers, TG1 isoverexpressed (see, for example, Friedrich et al. (1999) Histochem J.31(1):13-8).

Cancer, as used herein, refers to hyperproliferative conditions. Theterm denotes malignant as well as non-malignant cell populations. Suchdisorders have an excess cell proliferation of one or more subsets ofcells, which often appear to differ from the surrounding tissue bothmorphologically and genotypically. The excess cell proliferation can bedetermined by reference to the general population and/or by reference toa particular patient, e.g. at an earlier point in the patient's life.Hyperproliferative cell disorders can occur in different types ofanimals and in humans, and produce different physical manifestationsdepending upon the affected cells.

Cancers include leukemias, lymphomas (Hodgkins and non-Hodgkins),sarcomas, melanomas, adenomas, carcinomas of solid tissue includingbreast cancer and pancreatic cancer, hypoxic tumors, squamous cellcarcinomas of the mouth, throat, larynx, and lung, genitourinary cancerssuch as cervical and bladder cancer, hematopoietic cancers, head andneck cancers, and nervous system cancers, benign lesions such aspapillomas, and the like. Cancers that form solid tumors, i.e. otherthan leukemias and lymphomas, are of interest.

Cancer of particular interest are neurologic cancers, including braintumors. Neurologic tumors are classified according to the kind of cellfrom which the tumor seems to originate. Diffuse, fibrillaryastrocytomas are the most common type of primary brain tumor in adults.These tumors are divided histopathologically into three grades ofmalignancy: World Health Organization (WHO) grade II astrocytoma, WHOgrade III anaplastic astrocytoma and WHO grade IV glioblastomamultiforme (GBM). WHO grade II astocytomas are the most indolent of thediffuse astrocytoma spectrum. Astrocytomas display a remarkable tendencyto infiltrate the surrounding brain, confounding therapeutic attempts atlocal control. These invasive abilities are often apparent in low-gradeas well as high-grade tumors.

Glioblastoma multiforme is the most malignant stage of astrocytoma, withsurvival times of less than 2 years for most patients. Histologically,these tumors are characterized by high proliferation indices,endothelial proliferation and focal necrosis. The highly proliferativenature of these lesions likely results from multiple mitogenic effects.One of the hallmarks of GBM is endothelial proliferation. A host ofangiogenic growth factors and their receptors are found in GBMs.

There are biologic subsets of astrocytomas, which may reflect theclinical heterogeneity observed in these tumors. These subsets includebrain stem gliomas, which are a form of pediatric diffuse, fibrillaryastrocytoma that often follow a malignant course. Brain stem GBMs sharegenetic features with those adult GBMs that affect younger patients.Pleomorphic xanthoastrocytoma (PXA) is a superficial, low-gradeastrocytic tumor that predominantly affects young adults. While thesetumors have a bizarre histological appearance, they are typicallyslow-growing tumors that may be amenable to surgical cure. Some PXAs,however, may recur as GBM. Pilocytic astrocytoma is the most commonastrocytic tumor of childhood and differs clinically andhistopathologically from the diffuse, fibrillary astrocytoma thataffects adults. Pilocytic astrocytomas do not have the same genomicalterations as diffuse, fibrillary astrocytomas. Subependymal giant cellastrocytomas (SEGA) are periventricular, low-grade astrocytic tumorsthat are usually associated with tuberous sclerosis (TS), and arehistologically identical to the so-called “candle-gutterings” that linethe ventricles of TS patients. Similar to the other tumorous lesions inTS, these are slowly-growing and may be more akin to hamartomas thantrue neoplasms. Desmoplastic cerebral astrocytoma of infancy (DCAI) anddesmoplastic infantile ganglioglioma (DIGG) are large, superficial,usually cystic, benign astrocytomas that affect children in the firstyear or two of life.

Oligodendrogliomas and oligoastrocytomas (mixed gliomas) are diffuse,primarily CNS glial tumors that are clinically and biologically mostclosely related to the diffuse, fibrillary astrocytomas. The tumors,however, are far less common than astrocytomas and have generally betterprognoses than the diffuse astrocytomas. Oligodendrogliomas andoligoastrocytomas may progress, either to WHO grade III anaplasticoligodendroglioma or anaplastic oligoastrocytoma, or to WHO grade IVGBM. Thus, the genetic changes that lead to oligodendroglial tumorsconstitute yet another pathway to GBM.

Ependymomas are a clinically diverse group of gliomas that vary fromaggressive intraventricular tumors of children to benign spinal cordtumors in adults. Transitions of ependymoma to GBM are rare. Choroidplexus tumors are also a varied group of tumors that preferentiallyoccur in the ventricular system, ranging from aggressive supratentorialintraventricular tumors of children to benign cerebellopontine angletumors of adults. Choroid plexus tumors have been reported occasionallyin patients with Li-Fraumeni syndrome and von Hippel-Lindau (VHL)disease.

Medulloblastomas are malignant, primitive tumors that arise in theposterior fossa, primarily in children. Thjese tumors also occur inyoung adults. Medulloblastomas often are surgically resected withsubsequent treatment with chemotherapy and/or radiation. They may recurlocally or occasionally as drop metastasis from the posterior fossa tothe spine. Meningiomas are common intracranial tumors that arise in themeninges and compress the underlying brain. Although typicallyconsidered benign and only rarely frankly malignant, management of thesetumors often pose clinical challenges. Histological grades ofmeningiomas vary with the majority benign, WHO grade I/IV (82%); lesscommonly atypical, WHO II/IV (15%); and infrequently they occur asanaplastic or malignant, WHO grade III/IV (3%).

Schwannomas are benign tumors that arise on peripheral nerves.Schwannomas may arise on cranial nerves, particularly the vestibularportion of the eighth cranial nerve (vestibular schwannomas, acousticneuromas) where they present as cerebellopontine angle masses.Hemangioblastomas are tumors of uncertain origin that are composed ofendothelial cells, pericytes and so-called stromal cells. These benigntumors most frequently occur in the cerebellum and spinal cord of youngadults. Multiple hemangioblastomas are characteristic of vonHippel-Lindau disease (VHL). Hemangiopericytomas (HPCs) are dural tumorswhich may display locally aggressive behavior and may metastasize. Thehistogenesis of dural-based hemangiopericytoma (HPC) has long beendebated, with some authors classifying it as a distinct entity andothers classifying it as a subtype of meningioma.

The symptoms of both primary and metastatic brain tumors often depend onthe location in the brain and the size of the tumor. Since variousregions of the brain are responsible for specific functions, clinicalsymptoms will vary a great deal. Tumors in the frontal lobe of the brainmay cause weakness and paralysis, mood disturbances, difficultythinking, confusion and disorientation, and wide emotional mood swings.Parietal lobe tumors may cause seizures, numbness or paralysis,difficulty with handwriting, inability to perform simple mathematicalproblems, difficulty with certain movements, and loss of the sense oftouch. Tumors in the occipital lobe can cause loss of vision in half ofeach visual field, visual hallucinations, and seizures. Temporal lobetumors can cause seizures, perceptual and spatial disturbances, andreceptive aphasia. If a tumor occurs in the cerebellum, the person mayhave ataxia, loss of coordination, headaches, and vomiting. Tumors inthe hypothalamus may cause emotional changes, and changes in theperception of hot and cold. In addition, hypothalamic tumors may affectgrowth and nutrition in children. With the exception of the cerebellum,a tumor on one side of the brain causes symptoms and impairment on theopposite side of the body.

The compounds described herein are useful in the treatment ofindividuals suffering from the conditions described above, byadministering an effective dose of a tTGase inhibitor, through apharmaceutical formulation, and the like. Diagnosis of suitable patientsmay utilize a variety of criteria known to those of skill in the art.

Compounds of interest for inhibition of tTGase include those having thegeneral formulae

where R₁, R₂ and R₃ are independently selected from H, alkyl, alkenyl,cycloalkyl, aryl, heteroalkyl, heteroaryl, alkoxy, alkylthio, arakyl,aralkenyl, halo, haloalkyl, haloalkoxy, heterocyclyl, andheterocyclylalkyl groups. R₁ and R₂ can also be an amino acid, apeptide, a peptidomimetic, or a peptidic protecting groups.

Illustrative R₁ groups include Cbz, Fmoc, and Boc. In other embodimentsof the invention, R₁ is an arylether, aryl, alkylether or alkyl group,e.g. O-benzyl, benzyl, methyl or ethyl.

R₂ groups of interest include OMe, OtBu, Gly, and Gly-NH₂. In otherembodiments, R₂ is selected from the group consisting of (s)-Bn,(s)-CO₂Me, (s)-Me, (R)-Bn, (S)-CH₂CONHBn, (S)-(1H-inol-yl)-methyl, and(S)-(4-hydroxy-phenyl)-methyl.

R₃ is preferably a halo group, i.e. F, Cl, Br, and I, more preferably Clor Br.

X₁ and X₂ are selected from the group consisting of NH, O, and NR₄,where R₄ is a lower alkyl.

n is a whole number between 0 and 10, usually between 0 and 5, and moreusually between 0 and 3.

The tTGase inhibitory compounds of the invention from the isoxazoles canbe readily prepared using methods known in the art for other purposesand the teachings herein. Examples of synthetic routes to thesecompounds are also described in examples below For example, Castelhanoet al have demonstrated that the dihydroisoxazole derivative(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-phenyl-ethyl}-carbamicacid benzyl ester is an inhibitor of bovine epidermal transglutaminase(Castelhano et al., Bioorg. Chem. (1988) 16, 335-340; EP0237082).

A formula for transglutaminase inhibitors is:

In some embodiments, the inhibitor is an enantiopure isomer, having theformula:

In any of III, IIIa or IIIb, X is preferably a halo group, i.e. F, Cl,Br, and I.

R₁, R₂ and R₃ are independently selected from H, alkyl, alkenyl,cycloalkyl, aryl, heteroalkyl, heteroaryl, alkoxy, alkylthio, aralkyl,aralkenyl, halo, haloalkyl, haloalkoxy, heterocyclyl, andheterocyclylalkyl groups, or may also be an amino acid, a peptide, apeptidomimetic, or a peptidic protecting groups.

Illustrative R₁ groups include Cbz, Fmoc, and Boc. In other embodimentsof the invention, R₁ is an arylether, aryl, alkylether or alkyl group,e.g. O-benzyl, benzyl, methyl or ethyl.

R₂ groups of interest include OMe, OtBu, Gly, and Gly-NH₂. In otherembodiments, R₂ is selected from the group consisting of (S)-Bn,(S)-CO₂Me, (s)-Me, (R)-Bn, (S)- CH₂CONHBn, (S)-(1H-inol-yl)-methyl, and(S)-(4-hydroxy-phenyl)-methyl.

R₃ groups of interest include H, and cyclic alkyl or aryl groups.

In some embodiments of the invention, the tTGase inhibitor is as setforth in formula III, where X is Br, R₂ is as defined above, and mayinclude (S-),(R-)-4-fluoro-indole; (S-),(R-)-5-fluoro-indole;(S-),(R-)-6-fluoro-indole; (S-),(R-)-7-aza-indole; (S-)-p-hydroxyphenyl;(S-),(R-)-5-hydroxy-indole; (S-),(R-)-5-methoxy-indole;(S-)-5-fluoro-indole. R₁ is as defined above, and may specificallyinclude O-benzyl or 3-yl-O-quinoline. Such compounds may have thestructure:

and which may further include the chiral molecules:

where in any of IV, IVa and IVb, R₁, R₂ and R₃ are independentlyselected from H, alkyl, alkenyl, cycloalkyl, aryl, heteroalkyl,heteroaryl, alkoxy, alkylthio, arakyl, aralkenyl, halo, haloalkyl,haloalkoxy, heterocyclyl, and heterocyclylalkyl groups, and can also bean amino acid, a peptide, a peptidomimetic, or a peptidic protectinggroups.

Illustrative R₁ groups include arylether, cyclic alkylether,heteroarylether, particularly comprising N as a heteroatom.

R₂ groups of interest include substituted or unsubstituted heteroaryls,which include indoles substituted with one or more of Br, Cl, F, I,alkyl, particularly branched or unbranched lower alkyls of from one to 6carbons, OH, aza, and methoxy groups.

R₃ groups of interest include H, or R₂ and R₃ may form a cyclicheteroalkyl or heteroaryl group.

Here we identify new compounds within this genus that are especiallyeffective inhibitors of human tissue transglutaminase, and may thereforebe used to treat conditions such as Celiac Sprue, cancer, dermatitisherpetiformis, etc.

tTGase inhibitors of interest also include analogs of the dioxoindolineisatin. The cyclic α-keto amide structure of isatin serves as a goodanalog of γ-carboxamide group of tTGase glutamyl substrate. α-ketoamides are widely utilized as reversible inhibitors ofcysteine-dependent proteases and, in a similar way, the hetetocyclicstructure of isatin possesses an electrophilic carbonyl group whichcould be recognized by the enzyme as an analog of the substrateγ-carboxamide carbonyl group. Using standard procedures known in theart, the aromatic portion of the isatin structure can be derivatizedfurther to incorporate additional functional groups into the inhibitorsmimicking the other parts of peptide substrates.

The illustrative compounds of the invention described above were testedin a tTGase assay with recombinant human tissue transglutaminase, whichwas expressed, purified and assayed as described (Piper et al.,Biochemistry (2001) 41, 386-393). Competitive inhibition with respect tothe Cbz-Gln-Gly substrate was observed for all substrates; in all casesirreversible inactivation of the enzyme was also observed. Surprisingly,certain enantiomers were found to have low inhibition of tTGase invitro, but were potent chemosensitizers of tumor cells.

To facilitate an appreciation of the invention, the tTGase inhibitors ofthe invention have in part been described above with structurescontaining variable “R” groups that are defined by reference to thevarious organic moieties that can be present at the indicated positionin the structure. Below, brief definitions are provided for the phrasesused to define the organic moieties listed for each R group.

As used herein, “alkyl” refers to a straight or branched hydrocarbonchain radical consisting solely of carbon and hydrogen atoms, containingno unsaturation, having from one to eight carbon atoms, and which isattached to the rest of the molecule by a single bond, e.g., methyl,ethyl, n-propyl, 1-methylethyl(iso-propyl), n-butyl, n-pentyl,1,1-dimethylethyl(t-butyl), and the like. Unless stated otherwisespecifically in the specification, the alkyl radical may be optionallysubstituted by hydroxy, alkoxy, aryloxy, haloalkoxy, cyano, nitro,mercapto, alkylthio, —N(R⁸)₂, —C(O)OR⁸, —C(O)N(R⁸)₂ or —N(R⁸)C(O)R⁸where each R⁸ is independently hydrogen, alkyl, alkenyl, cycloalkyl,cycloalkylalkyl, aralkyl or aryl. Unless stated otherwise specificallyin the specification, it is understood that for radicals, as definedbelow, that contain a substituted alkyl group that the substitution canoccur on any carbon of the alkyl group.

“Alkoxy” refers to a radical of the formula —OR_(a) where R_(a) is analkyl radical as defined above, e.g., methoxy, ethoxy, n-propoxy,1-methylethoxy(iso-propoxy), n-butoxy, n-pentoxy,1,1-dimethylethoxy(t-butoxy), and the like. Unless stated otherwisespecifically in the specification, it is understood that for radicals,as defined below, that contain a substituted alkoxy group that thesubstitution can occur on any carbon of the alkoxy group. The alkylradical in the alkoxy radical may be optionally substituted as describedabove.

“Alkylthio” refers to a radical of the formula —SR_(a) where R_(a) is analkyl radical as defined above, e.g., methylthio, ethylthio,n-propylthio, 1-methylethylthio(iso-propylthio), n-butylthio,n-pentylthio, 1,1-dimethylethylthio(t-butylthio), and the like. Unlessstated otherwise specifically in the specification, it is understoodthat for radicals, as defined below, that contain a substitutedalkylthio group that the substitution can occur on any carbon of thealkylthio group. The alkyl radical in the alkylthio radical may beoptionally substituted as described above.

“Alkenyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, containing at least onedouble bond, having from two to eight carbon atoms, and which isattached to the rest of the molecule by a single bond or a double bond,e.g., ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl,and the like. Unless stated otherwise specifically in the specification,the alkenyl radical may be optionally substituted by hydroxy, alkoxy,haloalkoxy, cyano, nitro, mercapto, alkylthio, cycloalkyl, —N(R⁸)₂,—C(O)OR⁸, —C(O)N(R⁸)₂ or —N(R⁸)—C(O)—R⁸ where each R⁸ is independentlyhydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl.Unless stated otherwise specifically in the specification, it isunderstood that for radicals, as defined below, that contain asubstituted alkenyl group that the substitution can occur on any carbonof the alkenyl group.

“Aryl” refers to a phenyl or naphthyl radical. Unless stated otherwisespecifically in the specification, the term “aryl” or the prefix “ar-”(such as in “aralkyl”) is meant to include aryl radicals optionallysubstituted by one or more substituents selected from the groupconsisting of hydroxy, alkoxy, aryloxy, haloalkoxy, cyano, nitro,mercapto, alkylthio, cycloalkyl, —N(R⁸)₂, —C(O)OR⁸, —C(O)N(R⁸)₂ or—N(R⁸)C(O)R⁸ where each R⁸ is independently hydrogen, alkyl, alkenyl,cycloalkyl, cycloalkylalkyl, aralkyl or aryl. The term “aryl” alsorefers to the compound C₆H₅, i.e. Bn.

“Aralkyl” refers to a radical of the formula —R_(a)R_(b) where R_(a) isan alkyl radical as defined above and R_(b) is one or more aryl radicalsas defined above, e.g., benzyl, diphenylmethyl and the like. The arylradical(s) may be optionally substituted as described above.

“Aralkenyl” refers to a radical of the formula —R_(c)R_(b) where R_(c)is an alkenyl radical as defined above and R_(b) is one or more arylradicals as defined above, e.g., 3-phenylprop-1-enyl, and the like. Thearyl radical(s) and the alkenyl radical may be optionally substituted asdescribed above.

“Alkylene chain” refers to a straight or branched divalent hydrocarbonchain consisting solely of carbon and hydrogen, containing nounsaturation and having from one to eight carbon atoms, e.g., methylene,ethylene, propylene, n-butylene, and the like. The alkylene chain may beoptionally substituted by one or more substituents selected from thegroup consisting of aryl, halo, hydroxy, alkoxy, haloalkoxy, cyano,nitro, mercapto, alkylthio, cycloalkyl, —N(R⁸)₂, —C(O)OR⁸, —C(O)N(R⁸)₂or —N(R⁸)C(O)R⁸ where each R⁸ is independently hydrogen, alkyl, alkenyl,cycloalkyl, cycloalkylalkyl, aralkyl or aryl. The alkylene chain may beattached to the rest of the molecule through any two carbons within thechain.

“Alkenylene chain” refers to a straight or branched divalent hydrocarbonchain consisting solely of carbon and hydrogen, containing at least onedouble bond and having from two to eight carbon atoms, e.g., ethenylene,prop-1-enylene, but-1-enylene, pent-1-enylene, hexa-1,4-dienylene, andthe like. The alkenylene chain may be optionally substituted by one ormore substituents selected from the group consisting of aryl, halo,hydroxy, alkoxy, haloalkoxy, cyano, nitro, mercapto, alkylthio,cycloalkyl, —N(R⁸)₂, —C(O)OR⁸, —C(O)N(R⁸)₂ or —N(R⁸)C(O)R⁸ where each R⁸is independently hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl,aralkyl or aryl. The alkenylene chain may be attached to the rest of themolecule through any two carbons within the chain.

“Cycloalkyl” refers to a stable monovalent monocyclic or bicyclichydrocarbon radical consisting solely of carbon and hydrogen atoms,having from three to ten carbon atoms, and which is saturated andattached to the rest of the molecule by a single bond, e.g.,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, decalinyl and thelike. Unless otherwise stated specifically in the specification, theterm “cycloalkyl” is meant to include cycloalkyl radicals which areoptionally substituted by one or more substituents independentlyselected from the group consisting of alkyl, aryl, aralkyl, halo,haloalkyl, hydroxy, alkoxy, haloalkoxy, cyano, nitro, mercapto,alkylthio, cycloalkyl, —N(R⁸)₂, —C(O)OR⁸, —C(O)N(R⁸)₂ or —N(R⁸)C(O)R⁸where each R⁸ is independently hydrogen, alkyl, alkenyl, cycloalkyl,cycloalkylalkyl, aralkyl or aryl.

“Cycloalkylalkyl” refers to a radical of the formula —R_(a)R_(d) whereR_(a) is an alkyl radical as defined above and R_(d) is a cycloalkylradical as defined above. The alkyl radical and the cycloalkyl radicalmay be optionally substituted as defined above.

“Halo” refers to bromo, chloro, fluoro or iodo.

“Haloalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more halo radicals, as defined above, e.g.,trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl,1-fluoromethyl-2-fluoroethyl, 3-bromo-2-fluoropropyl,1-bromomethyl-2-bromoethyl, and the like.

“Haloalkoxy” refers to a radical of the formula —OR_(c) where R_(c) isan haloalkyl radical as defined above, e.g., trifluoromethoxy,difluoromethoxy, trichloromethoxy, 2,2,2-trifluoroethoxy,1-fluoromethyl-2-fluoroethoxy, 3-bromo-2-fluoropropoxy,1-bromomethyl-2-bromoethoxy, and the like.

“Heterocyclyl” refers to a stable 3- to 15-membered ring radical whichconsists of carbon atoms and from one to five heteroatoms selected fromthe group consisting of nitrogen, oxygen and sulfur. For purposes ofthis invention, the heterocyclyl radical may be a monocyclic, bicyclicor tricyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heterocyclylradical may be optionally oxidized; the nitrogen atom may be optionallyquaternized; and the heterocyclyl radical may be aromatic or partiallyor fully saturated. The heterocyclyl radical may not be attached to therest of the molecule at any heteroatom atom. Examples of suchheterocyclyl radicals include, but are not limited to, azepinyl,acridinyl, benzimidazolyl, benzthiazolyl, benzothiadiazolyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzotriazolyl, carbazolyl, cinnolinyl,decahydroisoquinolyl, dioxolanyl, furanyl, furanonyl, isothiazolyl,imidazolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, indolyl,indazolyl, isoindolyl, indolinyl, isoindolinyl, indolizinyl, isoxazolyl,isoxazolidinyl, morpholinyl, naphthyridinyl, oxadiazolyl,octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, oxazolyl,oxazolidinyl, oxiranyl, piperidinyl, piperazinyl, 4-piperidonyl,phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl,purinyl, pyrrolyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, pyridinyl,pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl,quinolinyl, quinuclidinyl, isoquinolinyl, thiazolyl, thiazolidinyl,thiadiazolyl, triazolyl, tetrazolyl, tetrahydrofuryl, triazinyl,tetrahydropyranyl, thienyl, thiamorpholinyl, thiamorpholinyl sulfoxide,and thiamorpholinyl sulfone. Unless stated otherwise specifically in thespecification, the term “heterocyclyl” is meant to include heterocyclylradicals as defined above which are optionally substituted by one ormore substituents selected from the group consisting of alkyl, halo,nitro, cyano, haloalkyl, haloalkoxy, aryl, heterocyclyl,heterocyclylalkyl, —OR⁸, —R⁷—OR⁸, —C(O)OR⁸, —R⁷—C(O)OR⁸, —C(O)N(R⁸)₂,—N(R⁸)₂, —R⁷—N(R⁸)₂, and —N(R⁸)C(O)R⁸ wherein each R⁷ is a straight orbranched alkylene or alkenylene chain and each R⁸ is independentlyhydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl.

“Heterocyclylalkyl” refers to a radical of the formula —R_(a)R_(e) whereR_(a) is an alkyl radical as defined above and R_(e) is a heterocyclylradical as defined above, and if the heterocyclyl is anitrogen-containing heterocyclyl, the heterocyclyl may be attached tothe alkyl radical at the nitrogen atom. The heterocyclyl radical may beoptionally substituted as defined above.

In the formulas provided herein, molecular variations are included,which may be based on isosteric replacement. “Isosteric replacement”refers to the concept of modifying chemicals through the replacement ofsingle atoms or entire functional groups with alternatives that havesimilar size, shape and electro-magnetic properties, e.g. O is theisosteric replacement of S, N, COON is the isosteric replacement oftetrazole, F is the isosteric replacement of H, sulfonate is theisosteric replacement of phosphate etc.

As used herein, compounds which are “commercially available” may beobtained from standard commercial sources including Acros Organics(Pittsburgh Pa.), Aldrich Chemical (Milwaukee Wis., including SigmaChemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), AvocadoResearch (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet(Cornwall, U.K.), Chemservice Inc. (West Chester Pa.), Crescent ChemicalCo. (Hauppauge N.Y.), Eastman Organic Chemicals, Eastman Kodak Company(Rochester N.Y.), Fisher Scientific Co. (Pittsburgh Pa.), FisonsChemicals (Leicestershire UK), Frontier Scientific (Logan Utah), ICNBiomedicals, Inc. (Costa Mesa Calif.), Key Organics (Cornwall U.K.),Lancaster Synthesis (Windham N.H.), Maybridge Chemical Co. Ltd.(Cornwall U.K.), Parish Chemical Co. (Orem Utah), Pfaltz & Bauer, Inc.(Waterbury Conn.), Polyorganix (Houston Tex.), Pierce Chemical Co.(Rockford Ill.), Riedel de Haen AG (Hannover, Germany), Spectrum QualityProduct, Inc. (New Brunswick, N.J.), TCI America (Portland Oreg.), TransWorld Chemicals, Inc. (Rockville Md.), Wako Chemicals USA, Inc.(Richmond Va.), Novabiochem and Argonaut Technology.

As used herein, “suitable conditions” for carrying out a synthetic stepare explicitly provided herein or may be discerned by reference topublications directed to methods used in synthetic organic chemistry.The reference books and treatise set forth above that detail thesynthesis of reactants useful in the preparation of compounds of thepresent invention, will also provide suitable conditions for carryingout a synthetic step according to the present invention.

As used herein, “methods known to one of ordinary skill in the art” maybe identified through various reference books and databases. Suitablereference books and treatises that detail the synthesis of reactantsuseful in the preparation of compounds of the present invention, orprovide references to articles that describe the preparation, includefor example, “Synthetic Organic Chemistry”, John Wiley & Sons, Inc., NewYork; S. R. Sandler et al., “Organic Functional Group Preparations,” 2ndEd., Academic Press, New York, 1983; H. O. House, “Modern SyntheticReactions”, 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L.Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, NewYork, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanismsand Structure”, 4th Ed., Wiley-Interscience, New York, 1992. Specificand analogous reactants may also be identified through the indices ofknown chemicals prepared by the Chemical Abstract Service of theAmerican Chemical Society, which are available in most public anduniversity libraries, as well as through on-line databases (the AmericanChemical Society, Washington, D.C., www.acs.org may be contacted formore details). Chemicals that are known but not commercially availablein catalogs may be prepared by custom chemical synthesis houses, wheremany of the standard chemical supply houses (e.g., those listed above)provide custom synthesis services.

“Optional” or “optionally” means that the subsequently described eventof circumstances may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. For example, “optionally substituted aryl” means that thearyl radical may or may not be substituted and that the descriptionincludes both substituted aryl radicals and aryl radicals having nosubstitution.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine,ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperazine, piperidine,N-ethylpiperidine, polyamine resins and the like. Particularly preferredorganic bases are isopropylamine, diethylamine, ethanolamine,trimethylamine, dicyclohexylamine, choline and caffeine.

The present invention provides the tTGase inhibitors in a variety offormulations for therapeutic administration. In one aspect, the agentsare formulated into pharmaceutical compositions by combination withappropriate, pharmaceutically acceptable carriers or diluents, and areformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. As such, administration of the tTGase inhibitors is achievedin various ways, although oral administration is a preferred route ofadministration. In some formulations, the tTGase inhibitors are systemicafter administration; in others, the inhibitor is localized by virtue ofthe formulation, such as the use of an implant that acts to retain theactive dose at the site of implantation.

In some pharmaceutical dosage forms, the tTGase inhibitors areadministered in the form of their pharmaceutically acceptable salts. Insome dosage forms, the tTGase inhibitor is used alone, while in others,the tTGase is used in combination with another pharmaceutically activecompounds.

In combination therapies for the treatment of Celiac Sprue and/orDermatitis Herpetiforms, the other active compound is, in someembodiments, a glutenase that can cleave or otherwise degrade a toxicgluten oligopeptide, as described in the Examples below.

In combination therapies for the treatment of cancer, the tTGaseinhibitor may be combined with a cytotoxic agent, or administered incombination with radiation therapy. Cytotoxic agents that act to reducecellular proliferation are known in the art and widely used. Such agentsinclude alkylating agents, such as nitrogen mustards, e.g.mechlorethamine, cyclophosphamide, melphalan (L-sarcolysin), etc.; andnitrosoureas, e.g. carmustine (BCNU), lomustine (CCNU), semustine(methyl-CCNU), streptozocin, chlorozotocin, etc.

Antimetabolite agents include pyrimidines, e.g. cytarabine (CYTOSAR-U),cytosine arabinoside, fluorouracil (5-FU), floxuridine (FUdR), etc.;purines, e.g. thioguanine (6-thioguanine), mercaptopurine (6-MP),pentostatin, fluorouracil (5-FU) etc.; and folic acid analogs, e.g.methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717),5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, etc.

Other natural products include azathioprine; brequinar; alkaloids andsynthetic or semi-synthetic derivatives thereof, e.g. vincristine,vinblastine, vinorelbine, etc.; podophyllotoxins, e.g. etoposide,teniposide, etc.; antibiotics, e.g. anthracycline, daunorubicinhydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin,doxorubicin, epirubicin and morpholino derivatives, etc.; phenoxizonebiscyclopeptides, e.g. dactinomycin; basic glycopeptides, e.g.bleomycin; anthraquinone glycosides, e.g. plicamycin (mithromycin);anthracenediones, e.g. mitoxantrone; azirinopyrrolo indolediones, e.g.mitomycin; and the like.

Other chemotherapeutic agents include metal complexes, e.g. cisplatin(cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; and hydrazines,e.g. N-methylhydrazine. Other anti-proliferative agents of interestinclude immunosuppressants, e.g. mycophenolic acid, thalidomide,desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF105685), etc.

The antineoplastic agents taxols (or taxanes) hyperstabilize polymerizedmicrotubules, leading to mitotic arrest and cytotoxicity inproliferating cells. Taxanes (or taxols), such as paclitaxel, docetaxel,etc. are of interest. Also of interest are the microtubule stabilizingepothilones (see Bollag et al. (1995) Cancer Research, Vol 55, Issue 112325-2333, herein incorporated by reference with respect to teachings ofthe class, and use thereof of these chemotherapeutic agents), e.g.epothilone A and epothilone B.

Retinoids, e.g. vitamin A, 13-cis-retinoic acid, trans-retinoic acid,isotretinoin, etc.; carotenoids, e.g. beta-carotene, vitamin D, etc.Retinoids regulate epithelial cell differentiation and proliferation,and are used in both treatment and prophylaxis of epithelialhyperproliferative disorders.

Topoisomerase inhibitors of interest include irinotecan (CPT-11), atopoisomerase I inhibitor. Other topoisomerase inhibitors of interest inthe subject methods include doxorubicin and carboplatinum, which inhibittype II topoisomerase.

In the such embodiments for the treatment of cancer, a cytotoxic agentmay be an alkylating agent such as BCNU or temozolomide, an antimitoticagent such as a taxane or epothilone, or arginine deiminase.

Pharmaceutical Formulations:

The tTGase inhibitors can be incorporated into a variety of formulationsfor therapeutic administration. In combination therapies, the tTGaseinhibitor and second agent can be delivered simultaneously, or within ashort period of time, by the same or by different routes. In oneembodiment of the invention, a co-formulation is used, where the twocomponents are combined in a single suspension. Alternatively, the twomay be separately formulated.

Part of the total dose may be administered by different routes. Suchadministration may use any route that results in systemic absorption, byany one of several known routes, including but not limited toinhalation, i.e. pulmonary aerosol administration; intranasal;sublingually; orally; and by injection, e.g. subcutaneously,intramuscularly, etc.

For injectables, the agents are used in formulations containingcyclodextrin, cremophor, DMSO, ethanol, propylene glycol, solutol,Tween, triglyceride and/or PEG. For oral preparations, the agents areused alone or in combination with appropriate additives to make tablets,powders, granules or capsules, for example, with conventional additives,such as lactose, mannitol, corn starch or potato starch; with binders,such as crystalline cellulose, cellulose derivatives, acacia, cornstarch or gelatins; with disintegrators, such as corn starch, potatostarch or sodium carboxymethylcellulose; with lubricants, such as talcor magnesium stearate; and in some embodiments, with diluents, bufferingagents, moistening agents, preservatives and flavoring agents.

In one embodiment of the invention, the oral formulations compriseenteric coatings, so that the active agent is delivered to theintestinal tract. Enteric formulations are often used to protect anactive ingredient from the strongly acid contents of the stomach. Suchformulations are created by coating a solid dosage form with a film of apolymer that is insoluble in acid environments and soluble in basicenvironments. Exemplary films are cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methylcellulose phthalate andhydroxypropyl methylcellulose acetate succinate, methacrylatecopolymers, and cellulose acetate phthalate.

Other enteric formulations of the tTGase inhibitors of the inventioncomprise engineered polymer microspheres made of biologically erodablepolymers, which display strong adhesive interactions withgastrointestinal mucus and cellular linings, can traverse both themucosal absorptive epithelium and the follicle-associated epitheliumcovering the lymphoid tissue of Peyer's patches. The polymers maintaincontact with intestinal epithelium for extended periods of time andactually penetrate it, through and between cells. See, for example,Mathiowitz et al. (1997) Nature 386 (6623): 410-414. Drug deliverysystems can also utilize a core of superporous hydrogels (SPH) and SPHcomposite (SPHC), as described by Dorkoosh et al. (2001) J ControlRelease 71(3):307-18.

In another embodiment, the tTGase inhibitor or formulation thereof isadmixed with food, or used to pre-treat foodstuffs containing glutens.

Formulations are typically provided in a unit dosage form, where theterm “unit dosage form,” refers to physically discrete units suitable asunitary dosages for human subjects, each unit containing a predeterminedquantity of tTGase inhibitor calculated in an amount sufficient toproduce the desired effect in association with a pharmaceuticallyacceptable diluent, carrier or vehicle. The specifications for the unitdosage forms of the present invention depend on the particular complexemployed and the effect to be achieved, and the pharmacodynamicsassociated with each complex in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Depending on the patient and condition being treated and on theadministration route, the tTGase inhibitor is administered in dosages of1 mg to 2000 mg/kg body weight per day, e.g. about 100, 500, 1000,10,000 mg/day for an average person. Durations of the regimen may befrom: 1×, 2× 3× daily; and in a combination regimen may be from about 1,about 7, about 14, etc. days prior to administration of second agent.Dosages are appropriately adjusted for pediatric formulation. Those ofskill will readily appreciate that dose levels can vary as a function ofthe specific inhibitor, the diet of the patient and the gluten contentof the diet, the severity of the symptoms, and the susceptibility of thesubject to side effects. Some of the inhibitors of the invention aremore potent than others. Preferred dosages for a given inhibitor arereadily determinable by those of skill in the art by a variety of means.A preferred means is to measure the physiological potency of a givencompound.

Various methods for administration are employed in the practice of theinvention. In one preferred embodiment, oral administration, for examplewith meals, is employed. The dosage of the therapeutic formulation canvary widely, depending upon the nature of the disease, the frequency ofadministration, the manner of administration, the clearance of the agentfrom the patient, and the like. The initial dose can be larger, followedby smaller maintenance doses. The dose can be administered asinfrequently as weekly or biweekly, or more often fractionated intosmaller doses and administered daily, with meals, semi-weekly, and thelike, to maintain an effective dosage level.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature), but someexperimental errors and deviations may be present. Unless indicatedotherwise, parts are parts by weight, molecular weight is weight averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

EXAMPLE 1 Synthesis of Dihydroxyisoxazole Containing tTGase Inhibitors

Synthesis of{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-phenyl-ethyl}-carbamicacid benzyl ester (n=0, X=NH, R₁=BnO, R₂=(S)-Bn, R₃=Br) (49).N-Cbz-L-Phe (0.30 g, 1.0 mmol) and HOBt (0.15 g, 1.1 eq) were dissolvedin 2 mL DMF. 3-Bromo-5-aminomethyl-4,5-dihydroisoazole (0.18 g, 1.0 eq),prepared following a reported procedure (Rohloff et al. (1992)Tetrahedron Lett. 33(22):3113-3116), was added to the solution cooled inan ice bath followed by EDCI (0.23 g, 1.2 eq). The ice bath was removedand the stirring was continued overnight. The solution was diluted withethyl acetate and washed with sat. NaHCO₃ solution and brine. Theorganic layer was dried over MgSO₄ and filtered. The solvent was removedby evaporation and the residue was purified by SiO₂ chromatography togive the title compound as a white solid (0.24 g, 52%).

¹H NMR (CDCl₃, 200 MHz): δ=7.34-7.26(m, 8H), 7.17(d, 2H, J=7.6 Hz),6.19-6.09(m, 1H), 5.21-5.15(m, 1H), 5.09(s, 2H), 4.74-4.60(m, 1H),4.41-4.36(m, 1H), 3.49-3.45(m, 2H), 3.26-3.12(m, 1H), 3.07(d, 2H, J=6.8Hz), 2.97-2.76(m, 1H) MS (ESI): m/z=460.1 [M+H]⁺, 482.2 [M+Na]⁺

Synthesis of(S)-2-Benzyloxycarbonylamino-4-[(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-butyricacid methyl ester (n=2, X=NH, R₁=BnO, R₂=(S)-CO₂Me, R₃=Br) (50). Thetitle compound was prepared according to the procedure for compound 49except using N-Cbz-L-Glu-OMe.

¹H NMR (CDCl₃, 200 MHz): δ=7.41-7.30(m, 5H), 6.22-6.12(m, 1H),5.63-5.57(m, 1H), 5.11(s, 2H), 4.82-4.74(m, 1H), 4.41-4.33(m, 1H),3.75(s, 3H), 3.54-3.48(m, 2H), 3.32-3.15(m, 1H), 3.02-2.88(m, 1H),2.34-2.22(m, 3H), 2.05-1.94(m, 1H) MS (ESI): m/z=456.1 [M+H]⁺, 478.2[M+Na]⁺

Synthesis of(S)-2-Benzyloxycarbonylamino-N-(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)-succinamicacid methyl ester (n=1, X=NH, R₁=BnO, R₂=(S)-CO₂Me, R₃=Br) (51). Thetitle compound was prepared according to the procedure for compound 49except using N-Cbz-L-Asp-OMe.

¹H NMR (CDCl₃, 200 MHz): δ=7.37-7.30(m, 5H), 6.00-5.90(m, 2H), 5.13(s,2H), 4.80-4.71(m, 1H), 4.63-4.58(m, 1H), 3.76(s, 3H), 3.54-3.44(m, 2H),3.33-3.23(m, 1H), 2.99-2.70(m, 3H) MS (ESI): m/z=442.1 [M+H]⁺, 464.2[M+Na]⁺

Synthesis of (S)-2-Benzyloxycarbonylamino-3-phenyl-propionic acid3-bromo-4,5-dihydro-isoxazol-5-ylmethyl ester (n=0, X=O, R₁=BnO,R₂=(S)-Bn, R₃=Br) (52). N-Cbz-L-Phe (0.30 g, 1.0 mmol) was dissolved inthe mixture of acetonitrile (6 mL), DIEA (0.18 mL, 1.0 eq) and excessallyl bromide (3 mL). After the reaction was allowed to proceedovernight, the reaction mixture was diluted with ethyl acetate, washedwith sat. Na₂CO₃ solution and brine, dried over MgSO₄ and concentratedto provide the ally ester as a clear oil (0.34 g, quant.). The ester(0.19 g, 0.57 mmol) and dibromoformaldoxime (0.14 g, 1.1 eq) weredissolved in 3 mL ethyl acetated and NaHCO₃ (0.21 g, 4.3 eq) was addedto the solution. The reaction mixture was stirred overnight, dilutedwith ethyl acetated and washed with sat. NaHCO₃ solution and brine. Theorganic layer was dried over MgSO₄ and the solvent was removed byevaporation. The residue was purified by SiO₂ chromatography to give thetitle compound as a white solid (0.15 g, 58%)

¹H NMR (CDCl₃, 200 MHz): δ=7.35-7.26(m, 8H), 7.16-7.14(m, 2H),5.20-5.05(m, 3H), 4.85-4.79(m, 1H), 4.68-4.63(m, 1H), 4.22-4.15(m, 2H),3.27-3.09(m, 3H), 2.96-2.77(m, 1H) MS (ESI): m/z=461.1 [M+H]⁺, 483.2[M+Na]⁺

Synthesis of{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-ethyl}-carbamicacid benzyl ester (n=0, X=NH, R₁=BnO, R₂=(S)-Me, R₃=Br) (53). The titlecompound was prepared according to the procedure for compound 49 exceptusing N-Cbz-L-Ala.

¹H NMR (CDCl₃, 200 MHz): δ=7.37-7.34(b, 5H), 6.68-6.45(m, 1H),5.24-5.18(m, 1H), 5.13(s, 2H), 4.80-4.76(m, 1H), 4.26-4.18(m, 1H),3.55-3.47(m, 2H), 3.33-3.19(m, 1H), 3.05-2.92(m, 1H), 1.39(d, 3H, J=7.0Hz) MS (ESI): m/z=384.1 [M+H]⁺, 406.1 [M+Na]⁺

Synthesis of(S)-2-Acetylamino-N-(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-phenyl-propionamide(n=0, X=NH, R₁=Me, R₂=(S)-Bn, R₃=Br) (54). The title compound wasprepared according to the procedure for compound 49 except usingN-Ac-L-Phe.

¹H NMR (CDCl₃, 200 MHz): δ=7.33-7.18(m, 5H), 6.14-6.09(m, 1H),6.02-5.97(m, 1H), 4.67-4.59(m, 2H), 3.49-3.41(m, 2H), 3.22-3.03(m, 3H),2.97-2.70(m, 1H), 2.00(s, 3H)

MS (ESI): m/z=368.1 [M+H]⁺, 390.2 [M+Na]⁺

Synthesis of{(R)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-phenyl-ethyl}-carbamicacid benzyl ester (n=0, X=NH, R₁=BnO, R₂=(R)-Bn, R₃=Br) (55). The titlecompound was prepared according to the procedure for compound 49 exceptusing N-Cbz-D-Phe.

¹H NMR (CDCl₃, 200 MHz): δ=7.34-7.26(m, 8H), 7.17(d, 2H, J=7.8 Hz),6.19-6.09(m, 1H), 5.21-5.15(m, 1H), 5.09(s, 2H), 4.74-4.60(m, 1H),4.41-4.36(m, 1H), 3.49-3.45(m, 2H), 3.26-3.12(m, 1H), 3.07(d, 2H, J=7.0Hz), 2.97-2.76(m, 1H) MS (ESI): m/z=460.1 [M+H]⁺, 482.2 [M+Na]⁺

Synthesis of{(S)-2-Benzylcarbamoyl-1-[(3-bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-ethyl}-carbamicacid benzyl ester (n=0, X=NH, R₁=BnO, R₂=(S)-CH₂CONHBn, R₃=Br) (56). Thetitle compound was prepared according to the procedure for compound 49except using β-benzylamide ofN-Cbz-L-Asp((S)-N-Benzyl-2-benzyloxycarbonylamino-succinamic acid).

¹H NMR (CDCl₃, 400 MHz): δ=7.38-7.24(m, 11H), 6.43-6.40(m, 1H),6.01-5.99(m, 1H), 5.14(s, 2H), 4.80-4.70(m, 1H), 4.58-4.52(m, 1H),4.41(d, 2H, J=6.4 Hz), 3.57-3.50(m, 2H), 3.25-3.12(m, 1H), 3.00-3.94(m,2H), 2.62-2.56(m, 1H)

MS (ESI): m/z=517.1 [M+H]⁺, 539.2 [M+Na]⁺

Synthesis of[(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(1H-indol-3-yl)-ethyl]-carbamicacid benzyl ester (n=0, X=NH, R₁=BnO, R₂=(S)-(1H-indol-3-yl)-methyl,R₃=Br) (57). The title compound was prepared according to the procedurefor compound 49 except using N-Cbz-L-Trp.

¹H NMR (CDCl₃, 400 MHz): δ=8.14(br, 1H), 7.70-7.63(m, 1H), 7.37-7.31(m,6H), 7.22-7.18(m, 1H), 7.13-7.09(m, 1H), 7.04-7.02(m, 1H), 6.15-6.10(m,1H), 5.45-5.39(m, 1H), 5.14-5.06(m, 2H), 4.59-4.47(m, 2H), 3.40-3.31(m,3H), 3.20-3.14(m, 1H), 3.11-3.04(m, 1H), 2.82-2.74(m, 1H)

MS (ESI): m/z=499.0 [M+H]⁺, 521.2 [M+Na]⁺

Synthesis of{(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-methyl-carbamoyl]-2-phenyl-ethyl}-carbamicacid benzyl ester (n=0, X=NMe, R₁=BnO, R₂=(S)-Bn, R₃=Br) (58). The titlecompound was prepared according to the procedure for compound 52 exceptusing N-methylallylamine.

¹H NMR (CDCl₃, 400 MHz): δ=7.34-7.26(m, 8H), 7.18-7.16(m, 2H),5.57-5.56(m, 1H), 5.12-5.05(m, 2H), 4.93-4.73(m, 2H), 3.80-3.67(m, 1H),3.36-3.17(m, 2H), 3.02-3.86(m, 6H)

MS (ESI): m/z=474.2 [M+H]⁺, 496.3 [M+Na]⁺

Synthesis of[(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-methyl-carbamoyl]-2-(4-hydroxy-phenyl)-ethyl]-carbamicacid benzyl ester (n=0, X=NH, R₁=BnO, R₂=(S)-(4-hydroxy-phenyl)-methyl,R₃=Br) (59). The title compound was prepared according to the procedurefor compound 49 except using N-Cbz-L-Tyr.

¹H NMR (DMSO-d₆, 400 MHz): δ=9.17(br, 1H), 8.27-8.23(m, 1H),7.43-7.40(m, 1H), 7.32-7.22(m, 5H), 7.03(d, 2H, J=7.6 Hz), 6.62(d, 2H,J=7.6 Hz), 4.93(s, 2H), 4.68-4.64(m, 1H), 4.13-4.11(m, 1H), 3.37-3.19(m,3H), 3.05-2.90(m, 1H), 2.81-2.77(m, 1H), 2.63-2.58(m, 1H)

MS (ESI): m/z=476.1 [M+H]⁺, 498.2 [M+Na]⁺

Synthesis of 1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-phenyl-urea(X₁=NH, X₂=NH, R₂=Ph, R₃=Br) (60).3-Bromo-5-aminomethyl-4,5-dihydroisoazole (20 mg, 0.11 mmol) and phenylisocyanate (13 uL, 1.0 eq) were dissolved in the mixture of THF (0.5 mL)and DMF (0.1 mL). After 30 min of stirring, the mixture was diluted withethyl acetate and washed with brine. The organic layer was dried overNa₂SO₄ and the solvents were removed by evaporation. The residue waspurified by SiO₂ chromatography to give the title compound.

¹H NMR (acetone-d₆, 400 MHz): δ=8.02(br, 1H), 7.50(d, 2H), 7.25-7.20(m,2H), 6.93(t, 1H), 6.24(br, 1H), 4.90-4.86(m, 1H), 3.56-3.54(m, 2H),3.48-3.41(m, 1H), 3.19-3.13(m, 1H)

MS (ESI): m/z=298.0 [M+H]⁺

Synthesis of1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-(2-chloro-5-trifluoromethyl-phenyl)-urea(X₁=NH, X₂=NH, R₂=2-chloro-5-trifluoromethyl-phenyl, R₃=Br) (61). Thetitle compound was prepared according to the procedure for compound 60except using 2-chloro-5-trifluoromethyl-phenylisocyanate

¹H NMR (acetone-d₆, 400 MHz): δ=8.82(s, 1H), 8.12(br, 1H), 7.62(d, 1H,J=8.0 Hz), 7.30(d, 1H, J=8.0 Hz), 6.90(br, 1H), 4.93-4.87(m, 1H),3.57-3.54(m, 2H), 3.49-3.42(m, 1H), 3.20-3.14(m, 1H)

MS (ESI): m/z=400.0 [M+H]⁺

Synthesis of1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-(4-chloro-2-trifluoromethyl-phenyl)-urea(X₁=NH, X₂=NH, R₂=4-chloro-2-trifluoromethyl-phenyl, R₃=Br) (62). Thetitle compound was prepared according to the procedure for compound 60except using 4-chloro-2-trifluoromethyl-phenylisocyanate.

¹H NMR (acetone-d₆, 400 MHz): δ=8.20(d, 1H, J=7.6 Hz), 7.66(br, 1H),7.62-7.60(m, 2H), 6.82(br, 1H), 4.89-4.85(m, 1H), 3.55-3.51(m, 2H),3.47-3.40(m, 1H), 3.18-3.12(m, 1H)

MS (ESI): m/z=400.0 [M+H]⁺

Synthesis of1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-(4-fluoro-phenyl)-urea(X₁=NH, X₂=NH, R₂=4-fluoro-phenyl, R₃=Br) (63). The title compound wasprepared according to the procedure for compound 60 except using4-fluoro-phenylisocyanate.

¹H NMR (acetone-d₆, 200 MHz): δ=8.06(br, 1H), 7.47-7.40(m, 2H),6.99-6.90(m, 2H), 5.94(br, 1H), 4.82-4.76(m, 1H), 3.45-3.30(m, 3H),3.17-3.04(m, 1H)

MS (ESI): m/z=316.0 [M+H]⁺

Synthesis of1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-(2,5-dimethyl-phenyl)-urea(X₁=NH, X₂=NH, R₂=2,5-dimethyl-phenyl, R₃=Br) (64). The title compoundwas prepared according to the procedure for compound 60 except using2,5-dimethyl-phenylisocyanate.

MS (ESI): m/z=326.0 [M+H]⁺

EXAMPLE 2 Synthesis of Dioxoindole Containing tTGase Inhibitors

Synthesis of 2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic acidpropylamide. 2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonyl chloride (0.10g, 0.41 mmol), prepared by the reaction of the sodium salt of5-isatinsulfonic acid with POCl₃, was dissolved in 5 mL THF. Thissolution was cooled in an ice bath and DIEA (0.14 mL, 2.0 eq) was addedslowly, followed by n-propylamine (35 uL, 1.0 eq). Stirring wascontinued for 40 min and the solution was diluted with ethyl acetate andwashed with brine. The organic layer was dried over Na₂SO₄ and thesolvent was removed by evaporation. The residue was purified by SiO₂chromatography to give the title compound (65 mg, 60%).

¹H NMR (CD₃CN, 400 MHz): δ=9.17(br, 1H), 8.02(d, 1H, J=8.0 Hz), 7.93(s,1H), 7.13(d, 1H, J=8.0 Hz), 5.62-5.58 (m, 1H), 2.85-2.80(m, 2H),1.48-1.42(m, 2H), 0.85(t, 3H, J=7.2 Hz)

MS (ESI): m/z=−267.1 [M−H]⁻

Synthesis of 2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic acidbenzylamide. The title compound was prepared from benzyl amine followingthe procedure for 2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic acidpropylamide.

¹H NMR (CD₃CN, 400 MHz): δ=9.19(br, 1H), 7.98(d, 1H, J=8.4 Hz), 7.85(s,1H), 7.31-7.21(m, 5H), 7.07(d, 1H, J=8.4 Hz), 6.11(t, 1H, J=6.3 Hz),4.11(d, 2H, J=6.3 Hz)

MS (ESI): m/z=−315.2 [M−H]⁻

Synthesis of(S)-1-(2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonyl)-pyrrolidine-2-carboxylicacid methyl ester. The title compound was prepared from L-Pro-OMefollowing the procedure for 2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonicacid propylamide.

¹H NMR (CDCl₃, 200 MHz): δ=8.85(br, 1H), 8.15-8.11 (m, 2H), 7.11(d, 1H,J=8.8 Hz), 4.47-4.41(m, 1H), 3.74(s, 3H), 3.45-3.39(m, 2H), 2.20-1.94(m, 4H)

MS (ESI): m/z=338.9[M+H]⁺

Synthesis of(S)-2-(2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonylamino)-3-phenyl-propionamide.The title compound was prepared from L-Phe-NH₂ following the procedurefor 2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic acid propylamide.

¹H NMR (CD₃CN, 200 MHz): δ=10.70(br, 1H), 7.78(d, 1H, J=8.4 Hz), 7.64(s,1H), 7.15-7.06(m, 6H), 6.90(d, 1H, J=8.4 Hz), 6.79(br, 1H), 6.08(br,1H), 3.98-3.87(m, 1H), 3.04-2.95(m, 1H), 2.76-2.64(m, 1H)

MS (ESI): m/z=−372.2 [M−H]⁻

Synthesis of(S)-N-(2-Dimethylamino-ethyl)-2-(2,3-dioxo-2,3-dihydro-1H-indole-5-sulfonylamino)-3-phenyl-propionamide.The title compound was prepared from L-Phe-NHCH₂CH₂NMe₂ following theprocedure for 2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic acidpropylamide.

¹H NMR (CD₃CN, 400 MHz): δ=7.84(d, 1H, J=8.0 Hz), 7.69(s, 1H),7.22-7.12(m, 6H), 6.98(d, 1H, J=8.0 Hz), 6.76(br, 1H), 3.96-3.93(m, 1H),3.10-3.02(m, 2H), 3.00-2.95(m, 1H), 2.78-2.72(m, 1H), 2.22-2.17(m, 2H),2.15(s, 6H)

MS (ESI): m/z=445.2 [M+H]⁺

Synthesis of 6-Bromo-7-methyl-1H-indole-2,3-dione. Chloral alcoholate(0.43 g, 1.05 eq) and Na₂SO₄(2.84 g, 20 mmol) were dissolved in 10 mLwater. 3-Bromo-2-methylaniline (0.33 g, 1.77 mmol) was added to thesolution followed by 0.16 mL conc. HCl aqueous solution and NH₂OH.HCl(0.38 g, 3.0 eq). The mixture was refluxed for 15 min and stirring wascontinued for additional 1 hr at RT. The precipitate was collected byfiltration, washed with water and dried under vacuum. This precipitatewas dissolved in 1 mL H₂SO₄ and the solution was heated (80° C.) for 15min. After cooling down to RT, the mixture was poured into ice-watermixture and the precipitate was collected, washed with water and driedunder vacuum to give the title compound (0.26 g, 61%).

¹H NMR (CD₃CN, 200 MHz): δ=9.02(BR, 1 h), 7.38(d, 1H, J=7.8 Hz), 7.30(d,1H, J=7.8 Hz), 2.30(s, 3H)

MS (ESI): m/z=−238.2 [M−H]⁻

Synthesis of 7-Methyl-6-phenyl-1H-indole-2,3-dione.6-Bromo-7-methyl-1H-indole-2,3-dione (100 mg, 0.38 mmol) andphenylboronic acid (53 mg, 1.1 eq) were dissolved in 10 mL DME.Pd(PPh₃)₄ (22 mg, 0.05 eq) were added followed by NaHCO₃ (65 mg, 2.0 eq)dissolved in 10 mL water. The mixture was refluxed for 2.5 hr and theorganic solvent was removed by evaporation. The mixture was extractedwith ethyl acetate and the combined organic layers were dried overNa₂SO₄ and purified by SiO₂ chromatography to give the title compound(50 mg, 51%).

¹H NMR (CDCl₃, 400 MHz): δ=8.53(br, 1H), 7.52(d, 1H, J=7.6 Hz),7.49-7.43(m, 3H), 7.31(d, 2H, J=6.4 Hz), 7.03(d, 1H, J=7.6 Hz), 2.16(s,3H)

MS (ESI): m/z=−236.3 [M−H]⁻

Inhibition of tTG. tTG (9 μM) was inactivated in 200 mM MOPS, pH=7.1, 5mM CaCl₂, 1 mM ETDA at 30° C. containing 0-600 μMPro-Gln-Pro-Aci-Leu-Pro-Tyr. Every 20 minutes a 40 μl aliquot wasremoved and residual tTG activity was assayed in 0.5 ml reactioncontaining 200 mM MOPS, pH=7.1, 5 mM CaCl₂, 1 mM ETDA, 10 mMα-ketoglutarate, 180 U/ml glutamate dehydrogenase (Biozyme laboratories)at 30° C. for 20 minutes by measuring the decrease of absorption at 340nm. Residual activity was corrected by the corresponding uninhibited tTGreaction (0 μM inhibitor) and fitted to an exponential decay. Kineticparameters were obtained by double-reciprocal plotting of the apparentsecond-order inactivation constant or, for isatin analogs, by fittingthe data for reversible inhibitors to a standard Michaelis Mentenequation with a competitive inhibition constant. The results of theseinhibition experiments are shown in Tables 1 and 2 below.

TABLE 1 Tissue transglutaminase inhibition by dihydroisoxazoles TestedCompound K_(l) (M) k_(inh) (min⁻¹) k_(inh)/K_(l) (min⁻¹M⁻¹){(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5- 0.73 × 10⁻³ 1.4 1900ylmethyl)-carbamoyl]-2-phenyl-ethyl}-carbamic acid benzyl ester (49)(S)-2-Benzyloxycarbonylamino-4-[(3-bromo-4,5-  1.6 × 10⁻³ 0.32 200dihydro-isoxazol-5-ylmethyl)-carbamoyl]-butyric acid methyl ester (50)(S)-2-Benzyloxycarbonylamino-N-(3-bromo-4,5- 0.87 × 10⁻³ 0.43 490dihydro-isoxazol-5-ylmethyl)-succinamic acid methyl ester (51)(S)-2-Benzyloxycarbonylamino-3-phenyl-  1.3 × 10⁻³ 0.32 230 propionicacid 3-bromo-4,5-dihydro-isoxazol-5- ylmethyl ester (52){(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5- 0.91 × 10⁻³ 0.41 450ylmethyl)-carbamoyl]-ethyl}-carbamic acid benzyl ester (53)(S)-2-Acetylamino-N-(3-bromo-4,5-dihydro-  2.7 × 10⁻³ 0.60 220isoxazol-5-ylmethyl)-3-phenyl-propionamide (54){(R)-1-[(3-Bromo-4,5-dihydro-isoxazol-5- 0.31 × 10⁻³ 0.29 940ylmethyl)-carbamoyl]-2-phenyl-ethyl}-carbamic acid benzyl ester (55){(S)-2-Benzylcarbamoyl-1-[(3-bromo-4,5-dihydro- 0.24 × 10⁻³ 0.54 2300isoxazol-5-ylmethyl)-carbamoyl]-ethyl}-carbamic acid benzyl ester (56)[(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5- 0.31 × 10⁻³ 0.78 2500ylmethyl)-carbamoyl]-2-(1H-indol-3-yl)-ethyl]- carbamic acid benzylester (57) {(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5- 0.26 × 10⁻³ 0.19 730ylmethyl)-methyl-carbamoyl]-2-phenyl-ethyl}- carbamic acid benzyl ester(58) [(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5- 0.42 × 10⁻³ 0.86 2000ylmethyl)-methyl-carbamoyl]-2-(4-hydroxy- phenyl)-ethyl]-carbamic acidbenzyl ester (59) 1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-  1.1 ×10⁻³ 0.89 810 phenyl-urea (60)1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3- 0.91 × 10⁻³ 0.95 1000(2-chloro-5-trifluoromethyl-phenyl)-urea (61)1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-  1.3 × 10⁻³ 1.1 850(4-chloro-2-trifluoromethyl-phenyl)-urea (62)1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3-  1.3 × 10⁻³ 1.0 770(4-fluoro-phenyl)-urea (63)1-(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-3- 0.96 × 10⁻³ 0.97 1000(2,5-dimethyl-phenyl)-urea (64)

TABLE 2 Tissue transglutaminase inhibition by Istatin derivatives

Tested compound (R) K_(l) (M) R1 = R2 = R3 = H 8.6 × 10⁻⁴ R2 = R3 = H,R1 = NO₂ 4.8 × 10⁻⁵ R2 = R3 = H; R1 = I 2.2 × 10⁻⁵ R2 = R3 = H; R1 = F1.8 × 10⁻⁵ R1 = R2 = H; R3 = Ph   4 × 10⁻⁴ R1 = R3 = H; R2 = Ph 3.5 ×10⁻⁴

The above results demonstrate that the compounds tested have tTGaseinhibitory activity.

EXAMPLE 3

The following compounds were synthesized as additional examples ofanalogues of[(S)-1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-methyl-carbamoyl]-2-(4-hydroxy-phenyl)-ethyl]-carbamicacid benzyl ester, for example as set forth in structure I of thepresent invention. Compounds reported below are specific examples oftransglutaminase inhibitors of use to treat Celiac Sprue as well asother diseases associated with undesirable activity of transglutaminase.

TABLE 3 kinh Kl kinh/Kl R = (/min) (mM) (/Mmin)

(S-),(R-)-4-fluoro-indole (S-),(R-)-5-fluoro-indole(S-),(R-)-6-fluoro-indole (S-),(R-)-7-aza-indole ERW907A ERW903 ERW907BERW907C <1200 >1200 <1200 <1200

(S-)-p-hydroxyphenyl (S-),(R-)-5-hydroxy-indole(S-),(R-)-5-methoxy-indole (S-)-5-fluoro-indole ERW1041A ERW1041BERW1041D ERW1069 0.184 2.57 0.283 0.1314 0.0631 1.38 0.1725 0.0049 29001850 1600 26800

ERW1041E 2.856 0.9527 3000

ERW1045 <1200

The specific compounds that were synthesized are as follows:

-   ERW903    [1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(5-fluoro-1H-indol-3-yl)-ethyl]-carbamic    acid benzyl ester (65)-   ERW907A    [1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(4-fluoro-1H-indol-3-yl)-ethyl]-carbamic    acid benzyl ester (66)-   ERW907B    [1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(6-fluoro-1H-indol-3-yl)-ethyl]-carbamic    acid benzyl ester (67)-   ERW907C    [1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(1H-pyrrolo[2,3-b]pyridin-3-yl)-ethyl]-carbamic    acid benzyl ester (68)-   ERW1041A    [1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(4-hydroxy-phenyl)-ethyl]-carbamic    acid quinolin-3-ylmethyl ester (69)-   ERW1041B=[1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(5-hydroxy-1H-indol-3-yl)-ethyl]-carbamic    acid quinolin-3-ylmethyl ester (70)-   ERW1041D    [1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(5-methoxy-1H-indol-3-yl)-ethyl]-carbamic    acid quinolin-3-ylmethyl ester (71)-   ERW1041E    2-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-pyrrolidine-1-carboxylic    acid quinolin-3-ylmethyl ester (72)-   ERW1045    2-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2,3-dihydro-indole-1-carboxylic    acid benzyl ester (73)-   ERW1069    [1-[(3-Bromo-4,5-dihydro-isoxazol-5-ylmethyl)-carbamoyl]-2-(5-fluoro-1H-indol-3-yl)-ethyl]-carbamic    acid quinolin-3-ylmethyl ester (74)

Synthesis of compounds ERW903, 907A, B, and C and 1045 were synthesizedusing the methods of Choi et al, Chemistry & Biology, Volume 12, Issue4, Pages 469-475.

Synthesis of compounds ERW1041A, B, C, D, E, and 1069 were synthesizedusing the methods of Choi et al. Chemistry & Biology, Volume 12, Issue4, Pages 469-475, except the amino-acid amines are protected by aquinoline-containing carbamate. This quinoline-containing carbamate issynthesized by reducing 3-quinolylcarbaldehyde with sodium borohydridein THF followed by an aqueous workup and flash chromatography yielding3-quinolylcarbinol. para-Nitro-phenylchloroformate is reacted with3-quinolylcarbinol in methylene chloride and N-methyl morpholine to givea carbonate of 3-quinoline methanol and para-Nitro-phenol. Afterpurification by flash chromatography, this carbonate is reacted with themethyl ester of the amino acid in neat DMF to give the named compounds.

EXAMPLE 4 Use of tTGase Inhibitors for Treating Neurologic Cancers

Preliminary pharmacokinetic and pharmacodynamic studies in mice on aprototype inhibitor, KCC009((S)-[3-(4-hydroxyphenyl)-2-N-(phenylmethyloxycarbonyl)aminopropanoicacid N′-(3′-bromo-4′,5′-dihydro-5′-isoxalyl)methylamide) (59), suggestedthat the compound had reasonable oral bioavailability, a short serumhalf-life, efficient TG2 inhibition in small intestinal tissue, and lowtoxicity. In vitro treatment of glioblastoma cells with KCC009 promotedapoptosis and enhanced sensitivity to chemotherapeutic agents. Suchtreatment also resulted in changes in levels of proteins altered inglioblastomas that enhance survival and are involved with promotingresistance to chemotherapy and radiation therapy. These changes inproteins included markedly decreased levels of phosphorylated Akt,survivin, phosphorylated Bad, and phosphorylated glycogen synthetasekinase 3β; and increased levels of the pro-apoptotic BH3-only protein,Bim. In vivo studies with subcutaneous murine DBT glioblastoma tumorsrevealed that KCC009 showed excellent synergism withN,N′-bis(2-chloroethyl)-N-nitrosourea (BCNU, carmustine). Together,these findings warranted further investigation into the use of KCC009for treating neuro-oncological diseases.

To test whether therapeutic doses of KCC009 can modulate TG2 activityassociated with intracranial glioblastomas, DBT tumors were injectedintracranially in mice. As judged by MRI, the mice consistentlyestablished intracranial tumors after 7-10 days following injections,which, if untreated, caused death by 2-3 weeks. In an initialdose-finding experiment, cohorts of mice were administered 4 daily dosesof vehicle or KCC009 (12.5 mg/kg, 25 mg/kg and 50 mg/kg) starting on day11 after intra-cranial injections. Twenty-four hours later, the micewere sacrificed and tumors analyzed. Grossly tumor dissections revealedthat control mouse brains had the largest tumors, and theircontra-lateral hemispheres were either swollen or infiltrated withmicroscopic tumor cells. Mice that received 12.5 mg/kg or 25 mg/kg doseshad smaller tumors and reduced brain swelling. One mouse for the 50mg/kg dose did not have any gross tumor. Based on these findings a doseof 35 mg/kg was used for subsequent experiments in mice harboringintracranial DBT tumors.

To evaluate the activity of KCC009 as a chemosensitizer, mice harboringDBT tumors were treated either with 10 mg/kg BCNU alone (2 mice) orBCNU+35 mg/kg KCC009 (3 mice). As before, 4 daily drug doses wereadministered intraperitoneally starting on day 11. Tumors were harvestedfor immuno-histological analysis 24 h after the last dose. As seen inFIG. 1, tumors from KCC009-treated animals showed a decrease infibronectin staining overall and an apparent dramatic decrease in theextracellular matrix. The controls showed linear strands of fibronectinin the ECM while the KCC009 groups had clumps of intracellularfibronectin.

To verify that the observed differences between BCNU versus BCNU+KCC009treated mice were due to inhibition of tumor associated TG2, micetreated with four daily doses of BCNU alone or BCNU+50 mg/kg KCC009 weresacrificed either 15 min or 24 h after the last dose, and tumorassociated TG2 activity was measured. As summarized in Table 4, KCC009treatment led to significant suppression of TG2 activity after 15 min,whereas enzyme activity recovers fully by 24 h. Both uninhibited andinhibited activity levels are consistent with similar measurements inthe small intestinal mucosa (data not shown).

TABLE 4 Inhibition of intracranial tumor associated TG2 by KCC009. Timeafter TG2 activity- BCNU alone TG2 activity- BCNU + KCC009 drug dose(nmol/h/mg protein) (nmol/h/mg protein) 15 min 2.1 ± 0.2 (n = 4) 1.2 ±0.3 (n = 4) 24 h 2.2 ± 0.3 (n = 2) 2.1 ± 0.2 (n = 3)

In addition to evaluating the chemosensitizing potential of KCC009, thiscompound's potential as a radiosensitizer was also evaluated. For thisclonogenic assays were used to measure colony formation after a singleradiation treatment. The studies were performed in triplicate withvarying doses of XRT and number of cells plated. In brief, treatment ofDBT cells in culture with 0.5 mM KCC009 enhanced cell death in responseto 3.2 Gy of X-ray radiation by two orders of magnitude. Arepresentative photo of a clonogenic assay plate treated with X-rayalone compared with a plate pre-treated with 0.5 mM KCC009 plus X-ray isshown in FIG. 2.

To highlight the clinical relevance of TG2 inhibition among tumors ofneurological origin, TG2 activity was assessed in tissue samples fromastrocytomas (Grade III/IV and IV/IV) and meningiomas (Grades I/IV;II/IV; and III/IV). TG2 activity was measured from 50 μM thick sectionsof brain tumor specimens collected from the operating room. Eachspecimen was coded and stored in the Tumor Repository. Samples analyzedincluded 4 normal brain sample, 6 anaplastic astrocytomas (GradeIII/IV); 9 glioblastomas (Grade III/IV), 7 typical meningiomas (GradeI/IV); 5 atypical meningiomas (Grade II/IV); and 3 anaplasticmeningiomas (Grade III/IV). The results in FIG. 3 show thatastrocytomas, glioblastomas and especially meningiomas had elevated TG2activity and are therefore candidates for KCC009 therapy.

To verify the therapeutic utility of KCC009 against meningiomas,IOMM-Lee meningioma cells were treated with 20 Gy of radiation with andwithout KCC009. As shown in FIG. 4, KCC009 dramatically sensitizedmeningioma cells to radiation. Similar data was also obtained whenKCC009 was used in combination with BCNU. To demonstrate therelationship between TG2 inhibition and extracellular matrix assembly,immunoprecipitated fibronectin from the different cultures were stainedwith anti-TG2 antibodies via Western blot analysis. As seen in FIG. 5,cells treated with KCC009 showed a loss of association between TG2 andfibronectin.

Activity of KCC009 against melanomas: Melanomas are cancers of theneural crest-derived cells that provide pigmentation to skin and othertissues. No current treatments substantially enhance patient survivalonce metastasis has occurred. As shown in FIG. 6, B16 mouse melanomacells are sensitive to KCC009 alone and especially KCC009 and BCNU.

To demonstrate the utility of KCC009 in cancer in combination with otherchemotherapeutic agents, HCT-116, a human colon cancer cell line, wastreated with epothilone C (a member of a promising class of anti-mitoticagents) and/or KCC009. Incubation of HCT-116 with increasing levels ofKCC009 in the 1-100 μM range reduced the GI₅₀ for epothilone C by>2-fold.

EXAMPLE 5

Due to the absence of an appropriate animal model for Celiac Sprue, thetherapeutic benefit of TG2 inhibitors for this disease can only bevalidated in patients. Our goal is therefore to identify a safe, oralTG2 inhibitor that can block mucosal TG2 activity in the upper smallintestine of Celiac Sprue patients. Such an agent could be used in thecontext of a short (2 week) blinded crossover trial in Celiac Spruepatients to determine if TG2 inhibition protects a Celiac Sprue patientfrom gluten-induced malabsorption. From available data, KCC009 may beprecisely such a compound. As shown in FIG. 7, intra-peritonealadministration of KCC009 results in strong inhibition of TG2 activity inmice on a short timescale (˜1 h), but this activity recovers completelywithin 8 h. To test whether orally administered KCC009 can inhibitjejunal TG2 activity, adult male rats were dosed with 35 mg/kg KCC009 ina kneaded gluten vehicle. Animals were sacrificed 1 h afteradministration of either vehicle-gluten (n=3) or drug-gluten (n=4), andjejunal mucosa was harvested. In vehicle treated animals, jejunal TG2activity was 21±7 pmol/min/mg protein, whereas in KCC009 treated ratsTG2 activity of 5.5±3 pmol/min/mg protein was observed. Thus, a suitableoral formulation may therefore enable local delivery of KCC009 tointestinal mucosa.

EXAMPLE 6 Preparation and Evaluation of Enantiopure DihydroxyisoxazoleContaining tTGase Inhibitors

To determine the activity of stereoisomers of the dihydroisoxazolewarhead, enantiopure versions of the dihydroisoxazole warhead wereprepared by enzymatic chiral resolution, as described below, and coupledto several N-protected amino-acids.

The preparation of chiral dihydroisoxazoles shown in Table 5 wasachieved by [3+2] cycloaddition of dibromoformaldoxime withallylacetate. The racemic product was resolved into enantiomers bytreatment with Amano Lipase PS in phosphate buffer, pH7, containing 15%acetone. These reaction conditions selectively hydrolyze the ester borneon (R-) dihydroisoxazoles, leaving the (S-) enantiomer unchanged. The(R-) dihydroisoxazole alcohol is readily separated from the (S-)dihydroisoxazole ester by flash chromatography. Hydrolysis of the (S-)dihydroisoxazole ester in methanol/water/potassium carbonate affordedthe second enantiomer of the dihydroisoxazole. The alcohols yielded fromthe chiral resolution had optical rotations of −135.5° (S-isomer) and+111.4° (R-isomer), consistent with published literature. Both compoundswere converted to the corresponding amines by means of the Mitsunobureaction using tetra-chloro-phthalimide as nucleophile, followed byaminolysis with ethylenediamine in ethanol/acetonitrile/THF. Theenantiopure dihydroisoxazole 30 and its enantiomer were coupled toN-protected amino acids under the same conditions as before.

Inhibitors containing the (R-) isomer of the dihydroisoxazole showed noinhibition of recombinant human TG2 in the case of KCC009, ERW1041E andERW1041A, and very weak inhibition was observed in the case of the(R)-ERW1069. In contrast, as summarized in the Table 5 below, the(S)-isomers of the above compounds were potent inhibitors of human TG2.

Kl K_(inh) k_(inh)/Kl Cmpd (mM) (min⁻¹) (M⁻¹min⁻¹)

(S)-ERW1041A 0.030 0.139 4,600

(S)-ERW1069 0.0013 0.072 57,000

(S)-ERW1041E 0.011 0.110 10,300

(S)-KCC009 0.068 0.155 2,300

The above in vitro studies indicate that bromodihydroisoxazoles with(S-) Stereochemistry are active in in vitro TG2 inhibition assays, andbromodihydroisoxazoles with (R-) stereochemistry are not. We thereforeanticipated finding that the (S-) stereoisomer of KCC009 was more activein the synergistic apoptotic potentiation observed with BCNU.

However, the data have shown that the (R-) isomer is more potent inchemosensitization assays. Specifically, confocal microscopy offibronectin staining of murine glioblastoma cells in the presence of(R)-ERW1041A and (S)-ERW1041A showed that treatment with the (R-) isomerdisrupted fibronectin in the extracellular matrix and increased thesusceptibility of cells to chemotherapeutic, BCNU. In contrast cellstreated with the (S-) isomer show structured fibronectin in theextracellular matrix with much less disruption. Similar data has alsobeen obtained in the intracranial DBT glioblastoma mouse model describedin the earlier filing. Thus, the (R) isomer of the bromodihydroisoxazolewarhead is better suited for therapy of glioblastoma and other cancers,even though it is a less active inhibitor of TG2.

A second approach to synthesizing the enantiopure amides is thesynthesis of the allylamine-derived dihydroisoxazole followed by chiralresolution of the racemic product with penicilin acylase via variants ofthe methods of Waldmann et al. Chem. Commun., 1997, 1861; Angew. Chem.,Int. Ed. Engl., 1997, 36, 647 and J. Am. Chem. Soc., 1997, 38, 6702.

A third approach to enantiopure amide versions of these compounds isfrom resolution of chiral mandelic acid amides of the aminedihydroisoxazoles after the method of Castelhano et al., Bioorg. Chem.(1988) 16, 335-340.

EXAMPLE 7 Synthesis and Evaluation of Ac-Pro-DON-Leu-Pro-Phe-NH₂DON=6-diazo-5-oxo-norleucine

Earlier disclosures had reported the synthesis and evaluation of apromising peptidic inhibitor of human TG2,Ac-Pro-Gln-Pro-DON-Leu-Pro-Phe-NH₂. We now disclose a truncated analogthat is easier to synthesize and has greater activity.

C₃₃H₄₆N₈O₇

Exact Mass: 666.35

Mol. Wt.: 666.77

m/e: 666.35 (100.0%), 667.35 (38.9%), 668.36 (6.5%), 668.35 (2.5%),669.36 (1.3%)

C, 59.44; H, 6.95; N, 16.81; O, 16.80

Synthesis: Ac-PELPF-NH₂ was synthesized using standard Boc chemistrysolid phase peptide and dried over KOH. 30 mg (46.8 μmol) Ac-PELPF-NH₂was dissolved in 4 ml of dry THF and cooled in an ice bath. 8.2 μl (1.6eq) of 4-Methylmorpholine was added followed by 7.3 μl (1.2 eq) ofIsobutyl chloroformate and allowed to react for 5 minutes (adapted fromHausch et al., Chem. Biol. (2003) 10, 225-231). The reaction mixture wasadded dropwise to a saturated solution of Diazomethane in 20 ml of dryDiethyl ether which was prechilled in an ice bath. The solution wasallowed to react for 30 min on ice followed by 30 min at roomtemperature. The solvent was removed by evaporation and the residue waspurified by high pressure liquid chromatography (C18,water/acetonitrile+0.1M Triethylammonium bicarbonate gradient).

Characterization: Ac-Pro-DON-Leu-Pro-Phe-NH₂ was characterized by themethod of Hausch et al. (Hausch et al., Chem. Biol. (2003) 10, 225-231).K_(I)=60 nM, k_(i)=0.5 min⁻¹, k_(i)/K_(I)=8.3 μM min⁻¹.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. Moreover, due to biological functionalequivalency considerations, changes can be made in protein structurewithout affecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

What is claimed is:
 1. A pharmaceutical formulation comprising: aneffective dose of a tTGase inhibitor of the formula:

wherein R₁ is quinolin-3-ylmethyl ester and R₂ is a substituted orunsubstituted heterocyclyl or heterocyclylalkyl group; R₃ is Br; n is 0;and X is NH.
 2. The formulation according to claim 1, wherein saidtTGase inhibitor is a substantially enantiopure compound.
 3. Theformulation of claim 1, wherein R₂ is selected from pyrrolidine,(5-hydroxy-1H-indol-3-yl)-ethyl, and (5-methoxy-1H-indol-3-yl)-ethyl. 4.The formulation of claim 1, wherein the tTGase inhibitor is


5. The formulation of claim 1, wherein the tTGase inhibitor has thestructure

wherein R is selected from (S-),(R-)-5-hydroxy-indole and(S-),(R-)-5-methoxy-indole.